122A Volume 58 Number 5 2004

focal pointBY ALAN VAN ORDEN KEIR FOGARTY

AND JAEMYEONG JUNG

DEPARTMENT OF CHEMISTRY

COLORADO STATE UNIVERSITY

FORT COLLINS COLORADO 80523

FluorescenceFluctuation

Spectroscopy AComing of Age Story

INTRODUCTION

This article presents the comingof age story for a suite of ul-trasensitive fluorescence-

based chemical analysis techniquesknown collectively as f luorescencefluctuation spectroscopy (FFS) Themost prominent example of FFS isfluorescence correlation spectrosco-py (FCS) which was introduced inthe 1970s as a way to noninvasivelymonitor the dynamics of chemicallyreacting systems1 FCS is used to an-alyze the time-dependent fluctua-tions in the fluorescence signal ob-served from an open subvolume ofan analyte solution These fluctua-tions are caused by spontaneouschanges in the molecular propertiesof the system localized within theregion of the sample being probedThey result mainly from random dif-fusion of the analyte molecules intoand out of the probe region causingvariations in the number of mole-cules being probed from one mo-

ment to the next Chemical reactionsaffecting the diffusion rates or theoptical properties of the moleculesalso create fluctuations in the fluo-rescence signal FCS analysis allowsthe temporal characteristics of theseprocesses to be monitored withoutthe need to perturb the equilibriumstate of the system

Advances in FFS technology overthe last decade have led to wide-spread acceptance of these methodsin a variety of different fields 2

Where it was once only possible tomonitor thousands of molecules at atime fluorescence signals can nowbe readily detected from individualmolecules as they traverse the detec-tion region one at a time greatly en-hancing the performance of the tech-nique Furthermore new analysismethods have been developed thatgive access to a large variety of fluo-rescence characteristics In the pastmost FFS strategies were based onanalyzing the translational diffusionrates of the molecules Today a large

selection of FFS techniques areavailable that can assess character-istics such as the specific brightness(a parameter describing the averagefluorescence emission rate of thefluorophor) the fluorescence aniso-tropy the fluorescence lifetime thespectral characteristics and the pho-tochemical properties of the analytein addition to its diffusion propertiesWhen the system contains a mixtureof fluorophors exhibiting differentfluorescence properties FFS analysiscan reveal the relative concentrationsof the different species If a chemicalreaction is occurring that alters oneof the measured parameters then theequilibrium constant and rate param-eters of the reaction can be mea-sured All of this translates into apowerful set of capabilities for mon-itoring an enormous variety of mo-lecular processes Indeed thesemethods are lsquolsquocoming of agersquorsquo in thesense that more often that not FFSmethods are being used as maturechemical analysis tools for solving

APPLIED SPECTROSCOPY 123A

real-world problems in bioanalyticalchemistry molecular biology andmaterials science

In its early stages of developmentFFS was regarded mainly as an ob-ject of study in its own right Peoplewere working out the details of howbest to configure the experimentsand how to collect and analyze thedata They were also trying to deter-mine the types of systems that couldbe studied and the characteristics ofthe system that were accessibleMost of the practitioners of FFSwere experts in the design and im-plementation of the technique To besure this type of activity is still tak-ing place New ways of collectingand analyzing the fluorescence dataare still being discovered new waysto interface FFS with other analyticalmethods such as microchip electro-phoresis devices and cellular imag-ing techniques are being devisedand chemical and biological systemsof increasing complexity are becom-ing accessible to FFS analysis Butto an ever-increasing extent re-searchers with little or no expertisein the methodological aspects of FFSare using standardized user-friendlycommercially available FFS instru-mentation to solve problems in theirrespective disciplines This is thehallmark of a mature analytical tech-nique that has come of age

A good example of the level ofmaturity achieved by FFS in recentyears is in its application to drug dis-covery research3ndash6 Drug discoveryresearchers are trying to uncovernew compounds that bind in a highlyspecific manner to selected molecu-lar targets such as proteins or cellsurface receptors This is often doneby screening large libraries of com-pounds in hopes of identifying a se-lect few with the desired bindingcharacteristics much like searchingfor a needle in a haystack Stringentrequirements are placed on the ana-lytical techniques used to assess thedegree of binding An ideal tech-nique would be able to measure theequilibrium binding constant of eachcompound with high speed and pre-cision it would be able to performassays in homogenous liquid solu-

tions it would be sensitive enoughto analyze samples containing fluidvolumes of only a few microliters orless and analyte concentrations in thesub-nanomolar to sub-micromolarrange and it would be able to per-form many such assays in a short pe-riod of time As will be discussedmore fully below FFS possessesmany of these desired characteris-tics

This coming of age story will fo-cus on the historical development ofFFS and its modern application inthe drug discovery field We will dis-cuss techniques that are familiar to afairly broad readership such as FCSas well as some newer methods suchas fluorescence intensity distributionanalysis that are just starting to takehold An important theme that willemerge is that the modern manifes-tations of these techniques trace theirroots to the detection of single fluo-rescent molecules as they freely dif-fuse in liquid solution and to theability to analyze the properties of asystem one molecule at a time Theoriginal demonstration of these ca-pabilities occurred in 1990 with thework of Keller and co-workers7

Hence the modern manifestations ofFFS are still relatively new As willbe seen below these techniques havemade remarkable strides toward ma-turity in this relatively short time

BROWNIAN MOTION

If we were to trace the history ofFFS to its ultimate origins we wouldbe led to the discovery of Brownianmotionmdashthe observation that micro-scopic particles suspended in liquidsfidget about due to collisions withthe surrounding solvent moleculesBrownian motion is ubiquitous andit would be impossible to say whenor where in human history it wasfirst observed The following lines ofpoetry penned by Ben Johnson earlyin the 17th century suggest the in-triguing possibility that the lsquolsquomove-ment of dust by atomsrsquorsquo was a notionthat existed in the collective con-sciousness long before any reliabletheories about the nature of atomsand molecules had been devised

Consider this small dust here in theglass by atoms moved

Could you believe that this the bodywas of one that loved

And in his mistressrsquo flame playinglike a fly

Was turned to cinders by her eyeYes and in death as life unblessed

To have it expressedEven ashes of lovers find no rest8

Anyone who has witnessed the fitfuldance of suspended microparticlesor grieved for a lost love will im-mediately relate to this apt compar-ison But the first carefully docu-mented description of Brownian mo-tion would have to wait another twocenturies In 1827 the Scottish bot-anist Robert Brown trained his opti-cal microscope on a suspension ofpollen grains in water and observedthe phenomenon that bears his namein exquisite detail Brownian diffu-sion had no satisfactory explanationwithin the theories of liquids thatprevailed at that time Liquids wereconsidered to be continuous fluidsnot assemblages of discreet particlesWhatever popular notions had exist-ed about the movement of atoms hadgiven way to this more pragmaticapproach Brown and his colleaguesassumed that the particles must belsquolsquoaliversquorsquo in some sense that theywere moving about under their ownanimate force But all efforts tolsquolsquokillrsquorsquo the particles and halt theirmotion proved fruitless Decades lat-er when the kinetic molecular theorybecame the topic of the day peoplefinally began thinking of this diffu-sion phenomenon as another mani-festation of molecules in motionmdashaglimpse into the atomic and molec-ular realm

Albert Einstein published a seriesof seminal papers on the theoreticalanalysis of Brownian motion begin-ning in 19059 By treating liquid wa-ter as an ensemble of indivisible par-ticles (or more precisely water mol-ecules) Einstein showed thatBrownian diffusion can be explainedby the random thermal motion of thewater molecules Pollen grains be-have as if they are being batted aboutby an invisible force because thenumber of water molecules colliding

124A Volume 58 Number 5 2004

focal point

with the particles is in a constantstate of flux One of Einsteinrsquos keypredictions was that on average thedisplacement of a particle should beproportional to the square root of thediffusion time This prediction wasexperimentally verified by theFrench physical chemist Jean Perrinwho carried out a series of carefullydesigned optical microscopy experi-ments to directly observe the dis-placements of microscopic particlesin water9 Einsteinrsquos theory and Per-rinrsquos experiments provided the mostcompelling evidence then to be hadabout the reality of atoms and mol-ecules They also represent the firsttime that a fluctuating signalmdashtherandom variation in the spatial co-ordinates of a microscopic particlemdashwas analyzed to deduce the molec-ular scale properties of a system andhence can be seen as the very firstlsquolsquomolecular fluctuation analysisrsquorsquo In-deed we may consider Jean Perrinto be the original fluctuation spec-troscopist

DYNAMIC LIGHTSCATTERING

A major advance in our ability tomeasure the diffusion characteristicsof microscopic and sub-microscopicparticles occurred in the late 1960sand early 1970s with the develop-ment of dynamic light scattering(DLS)810 It is no coincidence thatthe advent of these methods fol-lowed closely behind the inventionof the laser When a laser beam ir-radiates a suspension of particlessome of the laser light is elasticallyscattered by the particles at anglesother than the angle of incidenceBecause laser light is coherent thescattered light from multiple parti-cles can interfere to produce varia-tions in the scattered light intensityat different scattering angles Diffu-sion of the particles causes local var-iations in the particle concentrationwhich gives rise to fluctuations inthe intensity distribution of the scat-tered light intensity profile In one ofits implementations DLS monitorsthe intensity fluctuations at a givenscattering angle and then uses a sta-tistical analysis technique referred to

as autocorrelation analysis to char-acterize the time dependence ofthese fluctuations In general auto-correlation analysis measures thetime dependence of a given set ofmeasurement values which in thecase of DLS refers to the photocur-rent (or photoelectron counts) J(t)from a photomultiplier tube as afunction of time This information iscontained in the autocorrelationfunction G(t) given by

T21G(t) 5 lim J(t)J(t 1 t) dtETTrarr` 2T2

5 ^J(0)J(t)amp (1)

where T is the total measurementtime and t is the lagtime The auto-correlation function compares thescattered light intensity measured ata certain time t with the intensitymeasured at a later time t 1 t Whent is small compared to t there is lit-tle time for the particles to diffusegiving rise to comparatively largevalues of G(t) As t increases rela-tive to t G(t) begins to decay finallyreaching a constant value at infinite-ly long lagtimes The rate of decayof G(t) vs t is characteristic of thediffusion rate of the particles Anal-ysis of this decay gives the averagediffusion rate of the particles whichin turn yields the diffusion coeffi-cient D according to the EinsteinndashSmoluchowski equation

^(Dx)2amp 5 2Dt (2)

where ^(Dx)2amp is the average squareparticle displacement Knowledge ofthe diffusion coefficient gives rise tothe effective hydrodynamic radiusRH of the particles according to theStokesndashEinstein equation

kTD 5 (3)

6phRH

where k is the Boltzmann constantT is the temperature and h is the sol-vent viscosity DLS is a mature tech-nique that has found widespread usein a variety of research settings It isan extremely important tool for de-termining a host of macromolecularproperties such as molecular sizeand shape molecular weight etc

FLUORESCENCECORRELATIONSPECTROSCOPY

Beginning in 1972 Elson Magdeand Webb published a series of pa-pers describing a fluorescence-basedanalogue of DLS which they re-ferred to as fluorescence correlationspectroscopy (FCS)111ndash13 Webb oneof the original participants in theseefforts recently presented a very in-formative history of these develop-ments14 The authors realized that us-ing fluorescence measurements toanalyze the particle motion impartsseveral key advantages over conven-tional DLS Fluorescence is morechemically selective than light scat-tering and allows greater flexibilityin studying the motion of specificanalytes Also fluorescence can bedetected from molecules that aremuch too small to be detected byDLS which allows one to character-ize the motion of small molecules aswell as large macromolecules Final-ly fluorescence analysis opens theway to characterizing chemically re-acting systems Chemical reactionsgenerally do not create a largeenough index of refraction change tobe studied by DLS However spon-taneous chemical reactions can cre-ate fluctuations in the molecular dif-fusion properties andor other fluo-rescence characteristics that couldconceivably be analyzed via fluores-cence detection with much greatersensitivity

Fluorescence correlation spectros-copy was accomplished by focusingan excitation laser beam into the an-alyte solution and then monitoringthe fluorescence generated from thelaser beamrsquos focal region Diffusionof fluorophors into and out of the fo-cal volume altered the local concen-tration of the fluorophors giving riseto spontaneous fluorescence intensityfluctuations that could be analyzed inmuch the same way as the DLS sig-nal By analogy to DLS FCS com-pares the fluorescence intensity mea-sured at time t with the intensitymeasured at a later time t 1 t av-eraged over all values of t This in-formation is contained in an auto-correlation function G(t) A deriva-

APPLIED SPECTROSCOPY 125A

tion of G(t) which assumes a singlecomponent solution and only consid-ers diffusion along the axial dimen-sions of the laser beam yields theequation

1 1G(t) 5 (4)1 2N 1 1 ttd

Note that G(t) decays with t from amaximum at t 5 0 ms The ampli-tude of G(t) depends on the averagenumber of molecules N occupyingthe observation volume (ie the oc-cupancy) and its width depends ontd the average time it takes for amolecule to diffuse through the ob-servation volume The relationshipof td to the molecular diffusion co-efficient is given by

2v0t 5 (5)d 4D

where v0 is the radius of the excita-tion laser beam at its focus Henceas is the case with DLS measuringthe decay rate of G(t) vs t gives thediffusion coefficient of the analyteand from thence its molecular size

Another important aspect of FCSis its ability to characterize chemi-cally reacting systems Spontaneousfluctuations in the local concentra-tion of an analyte can be caused bychemical reactions occurring underequilibrium conditions as well as bymolecular diffusion By characteriz-ing these fluctuations FCS can mea-sure equilibrium constants and reac-tion rate parameters without the needto perturb the system equilibriumElson Magde and Webb derived au-tocorrelation functions for a numberof different scenarios in which thelocal concentration of the analytewas fluctuating due to some type ofspontaneous chemical reaction111

The case that will interest us here iswhere a small ligand molecule bindsto a much larger receptor thus re-ducing the diffusion coefficient ofthe ligand If the ligandndashreceptorcomplex is stable on the time scaleof its transit time through the detec-tion region then the autocorrelationfunction represents a linear combi-nation of two single-component au-tocorrelation functions correspond-ing to the free and bound ligand

2 12N QO i i 1 21 1 tti51 di

G(t) 5 (6)2^Iamp

Here i represents the free or thebound ligand Qi is the molecularbrightness of species i (the averageemission rate of a single molecule ofspecies i) and ^Iamp is the time-aver-aged total signal intensity due to thetotal fluorescence plus the back-ground signal If the diffusion ratesof the different species are sufficient-ly resolved then the autocorrelationfunction can be analyzed to deter-mine the relative concentrations ofbound and unbound ligand provid-ing a measure of the equilibriumbinding constant

In spite of its successful demon-stration and all of its potential ad-vantages FCS was not practical toimplement in those early years DLShad the advantage of being able toanalyze the motion of many uncor-related particles at once through theinterference of multiple scatteredlight waves Since fluorescenceemission is incoherent fluorescencefrom multiple fluorophors does notproduce such effects The only ef-fective way to analyze molecularmotion by fluorescence is to monitorthe individual molecules themselvesThis was simply not possible in the1970s The tricks of the trade for de-tecting single molecules had notbeen invented For one thing the de-tection volume was much too largeOne of the main sources of back-ground noise in single molecule fluo-rescence spectroscopy is scatteringof the excitation laser by the solventSince the backscattered light inten-sity is proportional to the size of thedetection volume the smaller the de-tection volume the better In the earlydays of FCS detection volumes ofpicoliters or greater were employedwhereas modern FCS instrumentsutilize femtoliter-sized detection vol-umes This meant that the backscat-tered light intensity exceeded thefluorescence emission of an individ-ual molecule Another problem wasthat the light collection and detectionefficiencies were far too low to de-

tect the fluorescence emitted by asingle fluorophor One reason for thiswas that the ultrasensitive singlephoton counting detectors used inmodern FCS had yet to be devel-oped In order to detect any signal atall it was necessary to probe 103

to 104 fluorophors at a time resultingin a large average fluorescence sig-nal Long data acquisition times onthe order of tens of minutes to hourswere needed in order to average outthe signal from spurious noise sourc-es and allow the tiny variations inthe average fluorescence signal topeek through In retrospect it is re-ally quite remarkable that the tech-nique worked at all But given itspractical limitations it did not comeinto widespread use for a number ofyears Still the foundation for thedevelopment of FCS and other FFSanalysis methods into a powerful setof modern research tools had beenlaid

SINGLE MOLECULECONFOCAL MICROSCOPY

The crucial advance that revital-ized FCS and led to a suite of alter-native FFS techniques was the com-bination of FCS with single mole-cule confocal microscopy Riglerand co-workers were the first to rec-ognize that confocal microscopywhen used as a spectroscopic anal-ysis tool had great potential forovercoming many of the challengesoriginally encountered with the ear-lier versions of FCS The earliest pa-pers on this subject date back to199215ndash17 Beginning in 1994 Riglerand co-workers1819 and Zare and co-workers2021 demonstrated that con-focal microscopy could be used todirectly detect the fluorescence emit-ted by individual molecules as theydiffused through the microscopic fo-cal volume of the confocal micro-scope This was an important exten-sion of the original single moleculedetection studies reported by Kellerand co-workers7 With this discov-ery the lengthy signal averagingtimes that were needed to measurethe autocorrelation function becamea thing of the past The correlationfunction could be determined based

126A Volume 58 Number 5 2004

focal point

FIG 1 Schematic diagram of a single molecule confocal fluorescence microscope setup used for FFS analysis The inset shows aschematic of the confocal detection volume and a simulated diffusion path of a single molecule through this volume

on a relatively small number of sin-gle molecule fluorescence signalsdetected over a period of a few sec-onds The dramatic rise in the pub-lication rate of FCS related papersthat occurred after these dates atteststo the impact of these important dis-coveries A number of books and re-view articles on single moleculefluorescence detection in solutionand its application to FCS have beenpublished over the years that detailthese advances222ndash29

Confocal microscopy has been animportant biological imaging tool formany years30 Its intended purpose isto create micrometer resolution fluo-rescence images of biological speci-mens and other materials In FFSthe confocal microscope is usedmore as a chemical analysis tool foranalyzing extremely small sub-vol-umes of dilute solutions than as an

imaging device (Fig 1) although itshould be noted that intracellular im-aging is another important areawhere FFS has started making animpact FCS is normally done by fo-cusing an excitation laser beam to itsdiffraction limit using a high numer-ical aperture (NA) microscope objec-tive positioning the focal region intothe analyte solution and monitoringthe fluorescence generated fromwithin the focal volume over timeThe same objective lens also servesto collect fluorescence from the sam-ple an arrangement referred to asepi-illumination A small pinholepositioned at the image plane of theobjective (the position where the im-age comes into focus behind the rearaperture of the objective) acts as aspatial filter to block fluorescencegenerated outside the focal regionfrom reaching the detector thus en-

suring that only the fluorescencegenerated within the focal region canbe detected

The spatial distribution of the lightintensity within the laser beam focusserves as the detection volume Thesize of the detection volume can beestimated by assuming a cylindrical-ly shaped focal volume with radiusv0 and height 2z0 where z0 is theaxial radius of the focal volume v0

and z0 are related to the NA of theobjective the wavelength l of theexcitation light and the index of re-fraction n of the sample mediumaccording to the equations

122l 2nlv 5 z 5 (7)0 0 22middotNA (NA)

In an experiment that utilizes a 13NA objective a 5145-nm laserbeam as the excitation source and an

APPLIED SPECTROSCOPY 127A

aqueous medium (n 5 133) the re-sulting detection volume is 03femtoliters This extremely small de-tection volume is important for sev-eral reasons It suppresses the back-ground noise caused by backscatter-ing of the excitation laser beamthrough Raliegh and Raman scatter-ing processes it enables optical ex-citation of the fluorophors to theirsaturation point using a modest av-erage laser power (1 mW) it en-sures that the number of fluorophorsbeing probed at any given time issmall and it allows samples with ex-tremely small volumes (microlitersor less) to be analyzed

Other aspects of confocal micros-copy that are important for singlemolecule detection include the highcollection efficiency of the objectivelens (25 for a 13 NA oil-im-mersion objective) the high trans-mission efficiency of the opticalcomponents in the wavelength rangeof interest and an efficient singlephoton counting detector Modernsingle photon counting avalanchephotodiode modules are able to de-tect visible photons with 30 to 70quantum efficiency All in all col-lectiondetection efficiencies of 5 to10 are attainable with modern con-focal microscope setups Consider-ing that many fluorophors can emitup to 106 to 108 photons per second(prior to photobleaching) whenpumped near their optical saturationpoint this can lead to photodetectionrates that exceed 105 photons persecond per molecule albeit overbrief time periods

Modern FFS takes advantage ofthe fact that dilute solutions (sub-nanomolar to sub-micromolar) offluorophors exhibit large amplitudefluorescence intensity fluctuationswhen probed by single moleculeconfocal microscopy This allowsthe fluctuations to be characterizedin a matter of seconds rather thanthe tens of minutes to hours neededin the earlier days Large amplitudefluctuations arise because the aver-age number of fluorophors occupy-ing the detection volume (ie theoccupancy) is small compared to thedeviation from the average at any

given time Random diffusion offluorophors into and out of the de-tection volume ensures that the num-ber of fluorophors being probed isnever the same from one moment tothe next Consider for example ananalyte concentration of 1 nM Atthis concentration the average num-ber of fluorophors within a 1-fem-toliter detection volume is 06 mol-ecules This means that on averagethe occupancy fluctuates betweenzero and one corresponding to de-viations from the mean occupancy of06 and 04 respectively If the mi-croscope is properly configured forsingle molecule detection then thefluorescence signal will be charac-terized by lsquolsquoquietrsquorsquo periods duringwhich only background noise is ob-served punctuated by brief intenselsquolsquoburstsrsquorsquo of signal due to the pas-sage of a single molecule throughthe detection volume (see Fig 2a)The durations of the bursts are char-acteristic of the diffusion rate of themolecules with average burst dura-tions typically ranging from a fewtens of microseconds to a few mil-liseconds depending on the mole-culersquos diffusion rate

At fluorophor concentrations be-tween 10 and 100 nM the numberof molecules occupying the detec-tion volume and hence the fluores-cence signal varies about a certainmean value (see Fig 2b) The fluo-rescence data collected under theseconditions is still representative ofindividual molecule transits eventhough more than one molecule isbeing probed at a time The ampli-tude of the autocorrelation functiontaken under these conditions will bereduced due to the inverse relation-ship with the occupancy number (seeFig 2c) As the concentration is in-creased above 100 nM the devia-tion in the occupancy becomes smallcompared to the average fluores-cence signal and the detector startsto reach its saturation point This re-quires lowering the laser power thusreducing the molecular brightness ofthe fluorophors These two effectsplace an upper limit on the fluoro-phor concentration in FCS analysisAt the lower end of the concentra-

tion scale the lengthy time intervalbetween detected molecules be-comes a limiting factor as doesbackground radiation coming fromRaman scattering by the solvent Ingeneral FFS is useful for analyteconcentrations in the range of 01nM to 100 nM It is sometimespossible to attain lower detectionlimits by rapidly scanning the focalvolume of the laser beam relative tothe sample (or vice versa)31 This en-larges the effective detection vol-ume and hence the average molec-ular occupancy without introducingunwanted background radiation

MEASURING THEAUTOCORRELATIONFUNCTION

In confocal microscopy basedFCS single photon counting meth-ods are used to measure the autocor-relation function Experimentallythis is accomplished by accumulat-ing the detected photons into succes-sive time bins of duration Dt Thefluorescence intensity I(t) at anygiven time is equivalent to the num-ber of detected photons ni dividedby the time interval Dt correspond-ing to t 5 iDt The autocorrelationfunction for a given lagtime is cal-culated from Eq 8 after an appro-priately large number of time inter-vals have been accumulated32

2M2k M2k

G(t) 5 (M 2 k) n n nO Oi i1k i1 2i51 i51

(8)

Here M is the total number of timebins and k indicates the time inter-val corresponding to lagtime t 5kDt In practice it is often conve-nient to allow the lengths of the suc-cessive time intervals to vary Pho-tons are initially collected into timebins of a few nanoseconds durationSubsequent photons are then accu-mulated into time bins of increasing-ly longer durations ranging from tensof nanoseconds to seconds Thislsquolsquomultiple-taursquorsquo approach impartssensitivity to fluctuations over abroad range of time scales (nanosec-onds to tens of seconds) without re-quiring excessive data accumula-

128A Volume 58 Number 5 2004

focal point

larr

FIG 2 Time-dependent fluorescence photo-count data and autocorrelation functions ob-tained from static solutions of fluorescent Rho-damine 6G molecules being probed by a sin-gle molecule confocal fluorescence detectionexperiment (a) Fluorescence data (red) ob-tained from a dilute (sub-nM) solution of fluo-rophors Photon bursts from individual mole-cules are clearly resolved The data were re-corded by accumulating the detected photo-counts into successive time bins of 1-msduration (b) Fluorescence data (blue) ob-tained from a more concentrated (10 nM)solution of fluorophors The solution is tooconcentrated for single molecule bursts to beclearly differentiated from the overall fluores-cence (c) Autocorrelation functions typical ofa dilute solution (red) and a more concentrat-ed solution (blue) The solid diamonds are ex-perimental data points and the solid curvesrepresent fits to a modified version of Eq 4that takes into account the lsquolsquotriplet blinkingrsquorsquoeffect at early lagtimes

tions All of these operations can beperformed using a commercial digi-tal correlator available from a num-ber of vendors

APPLICATION OFFLUORESCENCECORRELATIONSPECTROSCOPY IN DRUGDISCOVERY

Conventional diffusional FCS isthe oldest and most widely practicedform of FFS It is an extremely im-portant technique in a large varietyof fields A perusal of the recentbook Fluorescence CorrelationSpectroscopy Theory and Practiceattests to this fact2 A prominent ex-ample of its many uses is its contri-bution to one of the most criticalsteps in the drug discovery pro-cessmdashassessing the binding affinityof the drug candidate for a specifictarget receptor This is done by mon-itoring the change in the diffusiontime of the ligand when it binds toits receptor as illustrated in Fig 3Drug candidates are often small syn-thetic organic molecules but theycan also be peptides or even largebiological macromolecules such asproteins or DNA aptamers One ofthe ways in which they perform theirfunction is by binding to a specificreceptor so as to inhibit its biologicalactivity or to elicit some other bio-

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

APPLIED SPECTROSCOPY 123A

real-world problems in bioanalyticalchemistry molecular biology andmaterials science

In its early stages of developmentFFS was regarded mainly as an ob-ject of study in its own right Peoplewere working out the details of howbest to configure the experimentsand how to collect and analyze thedata They were also trying to deter-mine the types of systems that couldbe studied and the characteristics ofthe system that were accessibleMost of the practitioners of FFSwere experts in the design and im-plementation of the technique To besure this type of activity is still tak-ing place New ways of collectingand analyzing the fluorescence dataare still being discovered new waysto interface FFS with other analyticalmethods such as microchip electro-phoresis devices and cellular imag-ing techniques are being devisedand chemical and biological systemsof increasing complexity are becom-ing accessible to FFS analysis Butto an ever-increasing extent re-searchers with little or no expertisein the methodological aspects of FFSare using standardized user-friendlycommercially available FFS instru-mentation to solve problems in theirrespective disciplines This is thehallmark of a mature analytical tech-nique that has come of age

A good example of the level ofmaturity achieved by FFS in recentyears is in its application to drug dis-covery research3ndash6 Drug discoveryresearchers are trying to uncovernew compounds that bind in a highlyspecific manner to selected molecu-lar targets such as proteins or cellsurface receptors This is often doneby screening large libraries of com-pounds in hopes of identifying a se-lect few with the desired bindingcharacteristics much like searchingfor a needle in a haystack Stringentrequirements are placed on the ana-lytical techniques used to assess thedegree of binding An ideal tech-nique would be able to measure theequilibrium binding constant of eachcompound with high speed and pre-cision it would be able to performassays in homogenous liquid solu-

tions it would be sensitive enoughto analyze samples containing fluidvolumes of only a few microliters orless and analyte concentrations in thesub-nanomolar to sub-micromolarrange and it would be able to per-form many such assays in a short pe-riod of time As will be discussedmore fully below FFS possessesmany of these desired characteris-tics

This coming of age story will fo-cus on the historical development ofFFS and its modern application inthe drug discovery field We will dis-cuss techniques that are familiar to afairly broad readership such as FCSas well as some newer methods suchas fluorescence intensity distributionanalysis that are just starting to takehold An important theme that willemerge is that the modern manifes-tations of these techniques trace theirroots to the detection of single fluo-rescent molecules as they freely dif-fuse in liquid solution and to theability to analyze the properties of asystem one molecule at a time Theoriginal demonstration of these ca-pabilities occurred in 1990 with thework of Keller and co-workers7

Hence the modern manifestations ofFFS are still relatively new As willbe seen below these techniques havemade remarkable strides toward ma-turity in this relatively short time

BROWNIAN MOTION

If we were to trace the history ofFFS to its ultimate origins we wouldbe led to the discovery of Brownianmotionmdashthe observation that micro-scopic particles suspended in liquidsfidget about due to collisions withthe surrounding solvent moleculesBrownian motion is ubiquitous andit would be impossible to say whenor where in human history it wasfirst observed The following lines ofpoetry penned by Ben Johnson earlyin the 17th century suggest the in-triguing possibility that the lsquolsquomove-ment of dust by atomsrsquorsquo was a notionthat existed in the collective con-sciousness long before any reliabletheories about the nature of atomsand molecules had been devised

Consider this small dust here in theglass by atoms moved

Could you believe that this the bodywas of one that loved

And in his mistressrsquo flame playinglike a fly

Was turned to cinders by her eyeYes and in death as life unblessed

To have it expressedEven ashes of lovers find no rest8

Anyone who has witnessed the fitfuldance of suspended microparticlesor grieved for a lost love will im-mediately relate to this apt compar-ison But the first carefully docu-mented description of Brownian mo-tion would have to wait another twocenturies In 1827 the Scottish bot-anist Robert Brown trained his opti-cal microscope on a suspension ofpollen grains in water and observedthe phenomenon that bears his namein exquisite detail Brownian diffu-sion had no satisfactory explanationwithin the theories of liquids thatprevailed at that time Liquids wereconsidered to be continuous fluidsnot assemblages of discreet particlesWhatever popular notions had exist-ed about the movement of atoms hadgiven way to this more pragmaticapproach Brown and his colleaguesassumed that the particles must belsquolsquoaliversquorsquo in some sense that theywere moving about under their ownanimate force But all efforts tolsquolsquokillrsquorsquo the particles and halt theirmotion proved fruitless Decades lat-er when the kinetic molecular theorybecame the topic of the day peoplefinally began thinking of this diffu-sion phenomenon as another mani-festation of molecules in motionmdashaglimpse into the atomic and molec-ular realm

Albert Einstein published a seriesof seminal papers on the theoreticalanalysis of Brownian motion begin-ning in 19059 By treating liquid wa-ter as an ensemble of indivisible par-ticles (or more precisely water mol-ecules) Einstein showed thatBrownian diffusion can be explainedby the random thermal motion of thewater molecules Pollen grains be-have as if they are being batted aboutby an invisible force because thenumber of water molecules colliding

124A Volume 58 Number 5 2004

focal point

with the particles is in a constantstate of flux One of Einsteinrsquos keypredictions was that on average thedisplacement of a particle should beproportional to the square root of thediffusion time This prediction wasexperimentally verified by theFrench physical chemist Jean Perrinwho carried out a series of carefullydesigned optical microscopy experi-ments to directly observe the dis-placements of microscopic particlesin water9 Einsteinrsquos theory and Per-rinrsquos experiments provided the mostcompelling evidence then to be hadabout the reality of atoms and mol-ecules They also represent the firsttime that a fluctuating signalmdashtherandom variation in the spatial co-ordinates of a microscopic particlemdashwas analyzed to deduce the molec-ular scale properties of a system andhence can be seen as the very firstlsquolsquomolecular fluctuation analysisrsquorsquo In-deed we may consider Jean Perrinto be the original fluctuation spec-troscopist

DYNAMIC LIGHTSCATTERING

A major advance in our ability tomeasure the diffusion characteristicsof microscopic and sub-microscopicparticles occurred in the late 1960sand early 1970s with the develop-ment of dynamic light scattering(DLS)810 It is no coincidence thatthe advent of these methods fol-lowed closely behind the inventionof the laser When a laser beam ir-radiates a suspension of particlessome of the laser light is elasticallyscattered by the particles at anglesother than the angle of incidenceBecause laser light is coherent thescattered light from multiple parti-cles can interfere to produce varia-tions in the scattered light intensityat different scattering angles Diffu-sion of the particles causes local var-iations in the particle concentrationwhich gives rise to fluctuations inthe intensity distribution of the scat-tered light intensity profile In one ofits implementations DLS monitorsthe intensity fluctuations at a givenscattering angle and then uses a sta-tistical analysis technique referred to

as autocorrelation analysis to char-acterize the time dependence ofthese fluctuations In general auto-correlation analysis measures thetime dependence of a given set ofmeasurement values which in thecase of DLS refers to the photocur-rent (or photoelectron counts) J(t)from a photomultiplier tube as afunction of time This information iscontained in the autocorrelationfunction G(t) given by

T21G(t) 5 lim J(t)J(t 1 t) dtETTrarr` 2T2

5 ^J(0)J(t)amp (1)

where T is the total measurementtime and t is the lagtime The auto-correlation function compares thescattered light intensity measured ata certain time t with the intensitymeasured at a later time t 1 t Whent is small compared to t there is lit-tle time for the particles to diffusegiving rise to comparatively largevalues of G(t) As t increases rela-tive to t G(t) begins to decay finallyreaching a constant value at infinite-ly long lagtimes The rate of decayof G(t) vs t is characteristic of thediffusion rate of the particles Anal-ysis of this decay gives the averagediffusion rate of the particles whichin turn yields the diffusion coeffi-cient D according to the EinsteinndashSmoluchowski equation

^(Dx)2amp 5 2Dt (2)

where ^(Dx)2amp is the average squareparticle displacement Knowledge ofthe diffusion coefficient gives rise tothe effective hydrodynamic radiusRH of the particles according to theStokesndashEinstein equation

kTD 5 (3)

6phRH

where k is the Boltzmann constantT is the temperature and h is the sol-vent viscosity DLS is a mature tech-nique that has found widespread usein a variety of research settings It isan extremely important tool for de-termining a host of macromolecularproperties such as molecular sizeand shape molecular weight etc

FLUORESCENCECORRELATIONSPECTROSCOPY

Beginning in 1972 Elson Magdeand Webb published a series of pa-pers describing a fluorescence-basedanalogue of DLS which they re-ferred to as fluorescence correlationspectroscopy (FCS)111ndash13 Webb oneof the original participants in theseefforts recently presented a very in-formative history of these develop-ments14 The authors realized that us-ing fluorescence measurements toanalyze the particle motion impartsseveral key advantages over conven-tional DLS Fluorescence is morechemically selective than light scat-tering and allows greater flexibilityin studying the motion of specificanalytes Also fluorescence can bedetected from molecules that aremuch too small to be detected byDLS which allows one to character-ize the motion of small molecules aswell as large macromolecules Final-ly fluorescence analysis opens theway to characterizing chemically re-acting systems Chemical reactionsgenerally do not create a largeenough index of refraction change tobe studied by DLS However spon-taneous chemical reactions can cre-ate fluctuations in the molecular dif-fusion properties andor other fluo-rescence characteristics that couldconceivably be analyzed via fluores-cence detection with much greatersensitivity

Fluorescence correlation spectros-copy was accomplished by focusingan excitation laser beam into the an-alyte solution and then monitoringthe fluorescence generated from thelaser beamrsquos focal region Diffusionof fluorophors into and out of the fo-cal volume altered the local concen-tration of the fluorophors giving riseto spontaneous fluorescence intensityfluctuations that could be analyzed inmuch the same way as the DLS sig-nal By analogy to DLS FCS com-pares the fluorescence intensity mea-sured at time t with the intensitymeasured at a later time t 1 t av-eraged over all values of t This in-formation is contained in an auto-correlation function G(t) A deriva-

APPLIED SPECTROSCOPY 125A

tion of G(t) which assumes a singlecomponent solution and only consid-ers diffusion along the axial dimen-sions of the laser beam yields theequation

1 1G(t) 5 (4)1 2N 1 1 ttd

Note that G(t) decays with t from amaximum at t 5 0 ms The ampli-tude of G(t) depends on the averagenumber of molecules N occupyingthe observation volume (ie the oc-cupancy) and its width depends ontd the average time it takes for amolecule to diffuse through the ob-servation volume The relationshipof td to the molecular diffusion co-efficient is given by

2v0t 5 (5)d 4D

where v0 is the radius of the excita-tion laser beam at its focus Henceas is the case with DLS measuringthe decay rate of G(t) vs t gives thediffusion coefficient of the analyteand from thence its molecular size

Another important aspect of FCSis its ability to characterize chemi-cally reacting systems Spontaneousfluctuations in the local concentra-tion of an analyte can be caused bychemical reactions occurring underequilibrium conditions as well as bymolecular diffusion By characteriz-ing these fluctuations FCS can mea-sure equilibrium constants and reac-tion rate parameters without the needto perturb the system equilibriumElson Magde and Webb derived au-tocorrelation functions for a numberof different scenarios in which thelocal concentration of the analytewas fluctuating due to some type ofspontaneous chemical reaction111

The case that will interest us here iswhere a small ligand molecule bindsto a much larger receptor thus re-ducing the diffusion coefficient ofthe ligand If the ligandndashreceptorcomplex is stable on the time scaleof its transit time through the detec-tion region then the autocorrelationfunction represents a linear combi-nation of two single-component au-tocorrelation functions correspond-ing to the free and bound ligand

2 12N QO i i 1 21 1 tti51 di

G(t) 5 (6)2^Iamp

Here i represents the free or thebound ligand Qi is the molecularbrightness of species i (the averageemission rate of a single molecule ofspecies i) and ^Iamp is the time-aver-aged total signal intensity due to thetotal fluorescence plus the back-ground signal If the diffusion ratesof the different species are sufficient-ly resolved then the autocorrelationfunction can be analyzed to deter-mine the relative concentrations ofbound and unbound ligand provid-ing a measure of the equilibriumbinding constant

In spite of its successful demon-stration and all of its potential ad-vantages FCS was not practical toimplement in those early years DLShad the advantage of being able toanalyze the motion of many uncor-related particles at once through theinterference of multiple scatteredlight waves Since fluorescenceemission is incoherent fluorescencefrom multiple fluorophors does notproduce such effects The only ef-fective way to analyze molecularmotion by fluorescence is to monitorthe individual molecules themselvesThis was simply not possible in the1970s The tricks of the trade for de-tecting single molecules had notbeen invented For one thing the de-tection volume was much too largeOne of the main sources of back-ground noise in single molecule fluo-rescence spectroscopy is scatteringof the excitation laser by the solventSince the backscattered light inten-sity is proportional to the size of thedetection volume the smaller the de-tection volume the better In the earlydays of FCS detection volumes ofpicoliters or greater were employedwhereas modern FCS instrumentsutilize femtoliter-sized detection vol-umes This meant that the backscat-tered light intensity exceeded thefluorescence emission of an individ-ual molecule Another problem wasthat the light collection and detectionefficiencies were far too low to de-

tect the fluorescence emitted by asingle fluorophor One reason for thiswas that the ultrasensitive singlephoton counting detectors used inmodern FCS had yet to be devel-oped In order to detect any signal atall it was necessary to probe 103

to 104 fluorophors at a time resultingin a large average fluorescence sig-nal Long data acquisition times onthe order of tens of minutes to hourswere needed in order to average outthe signal from spurious noise sourc-es and allow the tiny variations inthe average fluorescence signal topeek through In retrospect it is re-ally quite remarkable that the tech-nique worked at all But given itspractical limitations it did not comeinto widespread use for a number ofyears Still the foundation for thedevelopment of FCS and other FFSanalysis methods into a powerful setof modern research tools had beenlaid

SINGLE MOLECULECONFOCAL MICROSCOPY

The crucial advance that revital-ized FCS and led to a suite of alter-native FFS techniques was the com-bination of FCS with single mole-cule confocal microscopy Riglerand co-workers were the first to rec-ognize that confocal microscopywhen used as a spectroscopic anal-ysis tool had great potential forovercoming many of the challengesoriginally encountered with the ear-lier versions of FCS The earliest pa-pers on this subject date back to199215ndash17 Beginning in 1994 Riglerand co-workers1819 and Zare and co-workers2021 demonstrated that con-focal microscopy could be used todirectly detect the fluorescence emit-ted by individual molecules as theydiffused through the microscopic fo-cal volume of the confocal micro-scope This was an important exten-sion of the original single moleculedetection studies reported by Kellerand co-workers7 With this discov-ery the lengthy signal averagingtimes that were needed to measurethe autocorrelation function becamea thing of the past The correlationfunction could be determined based

126A Volume 58 Number 5 2004

focal point

FIG 1 Schematic diagram of a single molecule confocal fluorescence microscope setup used for FFS analysis The inset shows aschematic of the confocal detection volume and a simulated diffusion path of a single molecule through this volume

on a relatively small number of sin-gle molecule fluorescence signalsdetected over a period of a few sec-onds The dramatic rise in the pub-lication rate of FCS related papersthat occurred after these dates atteststo the impact of these important dis-coveries A number of books and re-view articles on single moleculefluorescence detection in solutionand its application to FCS have beenpublished over the years that detailthese advances222ndash29

Confocal microscopy has been animportant biological imaging tool formany years30 Its intended purpose isto create micrometer resolution fluo-rescence images of biological speci-mens and other materials In FFSthe confocal microscope is usedmore as a chemical analysis tool foranalyzing extremely small sub-vol-umes of dilute solutions than as an

imaging device (Fig 1) although itshould be noted that intracellular im-aging is another important areawhere FFS has started making animpact FCS is normally done by fo-cusing an excitation laser beam to itsdiffraction limit using a high numer-ical aperture (NA) microscope objec-tive positioning the focal region intothe analyte solution and monitoringthe fluorescence generated fromwithin the focal volume over timeThe same objective lens also servesto collect fluorescence from the sam-ple an arrangement referred to asepi-illumination A small pinholepositioned at the image plane of theobjective (the position where the im-age comes into focus behind the rearaperture of the objective) acts as aspatial filter to block fluorescencegenerated outside the focal regionfrom reaching the detector thus en-

suring that only the fluorescencegenerated within the focal region canbe detected

The spatial distribution of the lightintensity within the laser beam focusserves as the detection volume Thesize of the detection volume can beestimated by assuming a cylindrical-ly shaped focal volume with radiusv0 and height 2z0 where z0 is theaxial radius of the focal volume v0

and z0 are related to the NA of theobjective the wavelength l of theexcitation light and the index of re-fraction n of the sample mediumaccording to the equations

122l 2nlv 5 z 5 (7)0 0 22middotNA (NA)

In an experiment that utilizes a 13NA objective a 5145-nm laserbeam as the excitation source and an

APPLIED SPECTROSCOPY 127A

aqueous medium (n 5 133) the re-sulting detection volume is 03femtoliters This extremely small de-tection volume is important for sev-eral reasons It suppresses the back-ground noise caused by backscatter-ing of the excitation laser beamthrough Raliegh and Raman scatter-ing processes it enables optical ex-citation of the fluorophors to theirsaturation point using a modest av-erage laser power (1 mW) it en-sures that the number of fluorophorsbeing probed at any given time issmall and it allows samples with ex-tremely small volumes (microlitersor less) to be analyzed

Other aspects of confocal micros-copy that are important for singlemolecule detection include the highcollection efficiency of the objectivelens (25 for a 13 NA oil-im-mersion objective) the high trans-mission efficiency of the opticalcomponents in the wavelength rangeof interest and an efficient singlephoton counting detector Modernsingle photon counting avalanchephotodiode modules are able to de-tect visible photons with 30 to 70quantum efficiency All in all col-lectiondetection efficiencies of 5 to10 are attainable with modern con-focal microscope setups Consider-ing that many fluorophors can emitup to 106 to 108 photons per second(prior to photobleaching) whenpumped near their optical saturationpoint this can lead to photodetectionrates that exceed 105 photons persecond per molecule albeit overbrief time periods

Modern FFS takes advantage ofthe fact that dilute solutions (sub-nanomolar to sub-micromolar) offluorophors exhibit large amplitudefluorescence intensity fluctuationswhen probed by single moleculeconfocal microscopy This allowsthe fluctuations to be characterizedin a matter of seconds rather thanthe tens of minutes to hours neededin the earlier days Large amplitudefluctuations arise because the aver-age number of fluorophors occupy-ing the detection volume (ie theoccupancy) is small compared to thedeviation from the average at any

given time Random diffusion offluorophors into and out of the de-tection volume ensures that the num-ber of fluorophors being probed isnever the same from one moment tothe next Consider for example ananalyte concentration of 1 nM Atthis concentration the average num-ber of fluorophors within a 1-fem-toliter detection volume is 06 mol-ecules This means that on averagethe occupancy fluctuates betweenzero and one corresponding to de-viations from the mean occupancy of06 and 04 respectively If the mi-croscope is properly configured forsingle molecule detection then thefluorescence signal will be charac-terized by lsquolsquoquietrsquorsquo periods duringwhich only background noise is ob-served punctuated by brief intenselsquolsquoburstsrsquorsquo of signal due to the pas-sage of a single molecule throughthe detection volume (see Fig 2a)The durations of the bursts are char-acteristic of the diffusion rate of themolecules with average burst dura-tions typically ranging from a fewtens of microseconds to a few mil-liseconds depending on the mole-culersquos diffusion rate

At fluorophor concentrations be-tween 10 and 100 nM the numberof molecules occupying the detec-tion volume and hence the fluores-cence signal varies about a certainmean value (see Fig 2b) The fluo-rescence data collected under theseconditions is still representative ofindividual molecule transits eventhough more than one molecule isbeing probed at a time The ampli-tude of the autocorrelation functiontaken under these conditions will bereduced due to the inverse relation-ship with the occupancy number (seeFig 2c) As the concentration is in-creased above 100 nM the devia-tion in the occupancy becomes smallcompared to the average fluores-cence signal and the detector startsto reach its saturation point This re-quires lowering the laser power thusreducing the molecular brightness ofthe fluorophors These two effectsplace an upper limit on the fluoro-phor concentration in FCS analysisAt the lower end of the concentra-

tion scale the lengthy time intervalbetween detected molecules be-comes a limiting factor as doesbackground radiation coming fromRaman scattering by the solvent Ingeneral FFS is useful for analyteconcentrations in the range of 01nM to 100 nM It is sometimespossible to attain lower detectionlimits by rapidly scanning the focalvolume of the laser beam relative tothe sample (or vice versa)31 This en-larges the effective detection vol-ume and hence the average molec-ular occupancy without introducingunwanted background radiation

MEASURING THEAUTOCORRELATIONFUNCTION

In confocal microscopy basedFCS single photon counting meth-ods are used to measure the autocor-relation function Experimentallythis is accomplished by accumulat-ing the detected photons into succes-sive time bins of duration Dt Thefluorescence intensity I(t) at anygiven time is equivalent to the num-ber of detected photons ni dividedby the time interval Dt correspond-ing to t 5 iDt The autocorrelationfunction for a given lagtime is cal-culated from Eq 8 after an appro-priately large number of time inter-vals have been accumulated32

2M2k M2k

G(t) 5 (M 2 k) n n nO Oi i1k i1 2i51 i51

(8)

Here M is the total number of timebins and k indicates the time inter-val corresponding to lagtime t 5kDt In practice it is often conve-nient to allow the lengths of the suc-cessive time intervals to vary Pho-tons are initially collected into timebins of a few nanoseconds durationSubsequent photons are then accu-mulated into time bins of increasing-ly longer durations ranging from tensof nanoseconds to seconds Thislsquolsquomultiple-taursquorsquo approach impartssensitivity to fluctuations over abroad range of time scales (nanosec-onds to tens of seconds) without re-quiring excessive data accumula-

128A Volume 58 Number 5 2004

focal point

larr

FIG 2 Time-dependent fluorescence photo-count data and autocorrelation functions ob-tained from static solutions of fluorescent Rho-damine 6G molecules being probed by a sin-gle molecule confocal fluorescence detectionexperiment (a) Fluorescence data (red) ob-tained from a dilute (sub-nM) solution of fluo-rophors Photon bursts from individual mole-cules are clearly resolved The data were re-corded by accumulating the detected photo-counts into successive time bins of 1-msduration (b) Fluorescence data (blue) ob-tained from a more concentrated (10 nM)solution of fluorophors The solution is tooconcentrated for single molecule bursts to beclearly differentiated from the overall fluores-cence (c) Autocorrelation functions typical ofa dilute solution (red) and a more concentrat-ed solution (blue) The solid diamonds are ex-perimental data points and the solid curvesrepresent fits to a modified version of Eq 4that takes into account the lsquolsquotriplet blinkingrsquorsquoeffect at early lagtimes

tions All of these operations can beperformed using a commercial digi-tal correlator available from a num-ber of vendors

APPLICATION OFFLUORESCENCECORRELATIONSPECTROSCOPY IN DRUGDISCOVERY

Conventional diffusional FCS isthe oldest and most widely practicedform of FFS It is an extremely im-portant technique in a large varietyof fields A perusal of the recentbook Fluorescence CorrelationSpectroscopy Theory and Practiceattests to this fact2 A prominent ex-ample of its many uses is its contri-bution to one of the most criticalsteps in the drug discovery pro-cessmdashassessing the binding affinityof the drug candidate for a specifictarget receptor This is done by mon-itoring the change in the diffusiontime of the ligand when it binds toits receptor as illustrated in Fig 3Drug candidates are often small syn-thetic organic molecules but theycan also be peptides or even largebiological macromolecules such asproteins or DNA aptamers One ofthe ways in which they perform theirfunction is by binding to a specificreceptor so as to inhibit its biologicalactivity or to elicit some other bio-

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

124A Volume 58 Number 5 2004

focal point

with the particles is in a constantstate of flux One of Einsteinrsquos keypredictions was that on average thedisplacement of a particle should beproportional to the square root of thediffusion time This prediction wasexperimentally verified by theFrench physical chemist Jean Perrinwho carried out a series of carefullydesigned optical microscopy experi-ments to directly observe the dis-placements of microscopic particlesin water9 Einsteinrsquos theory and Per-rinrsquos experiments provided the mostcompelling evidence then to be hadabout the reality of atoms and mol-ecules They also represent the firsttime that a fluctuating signalmdashtherandom variation in the spatial co-ordinates of a microscopic particlemdashwas analyzed to deduce the molec-ular scale properties of a system andhence can be seen as the very firstlsquolsquomolecular fluctuation analysisrsquorsquo In-deed we may consider Jean Perrinto be the original fluctuation spec-troscopist

DYNAMIC LIGHTSCATTERING

A major advance in our ability tomeasure the diffusion characteristicsof microscopic and sub-microscopicparticles occurred in the late 1960sand early 1970s with the develop-ment of dynamic light scattering(DLS)810 It is no coincidence thatthe advent of these methods fol-lowed closely behind the inventionof the laser When a laser beam ir-radiates a suspension of particlessome of the laser light is elasticallyscattered by the particles at anglesother than the angle of incidenceBecause laser light is coherent thescattered light from multiple parti-cles can interfere to produce varia-tions in the scattered light intensityat different scattering angles Diffu-sion of the particles causes local var-iations in the particle concentrationwhich gives rise to fluctuations inthe intensity distribution of the scat-tered light intensity profile In one ofits implementations DLS monitorsthe intensity fluctuations at a givenscattering angle and then uses a sta-tistical analysis technique referred to

as autocorrelation analysis to char-acterize the time dependence ofthese fluctuations In general auto-correlation analysis measures thetime dependence of a given set ofmeasurement values which in thecase of DLS refers to the photocur-rent (or photoelectron counts) J(t)from a photomultiplier tube as afunction of time This information iscontained in the autocorrelationfunction G(t) given by

T21G(t) 5 lim J(t)J(t 1 t) dtETTrarr` 2T2

5 ^J(0)J(t)amp (1)

where T is the total measurementtime and t is the lagtime The auto-correlation function compares thescattered light intensity measured ata certain time t with the intensitymeasured at a later time t 1 t Whent is small compared to t there is lit-tle time for the particles to diffusegiving rise to comparatively largevalues of G(t) As t increases rela-tive to t G(t) begins to decay finallyreaching a constant value at infinite-ly long lagtimes The rate of decayof G(t) vs t is characteristic of thediffusion rate of the particles Anal-ysis of this decay gives the averagediffusion rate of the particles whichin turn yields the diffusion coeffi-cient D according to the EinsteinndashSmoluchowski equation

^(Dx)2amp 5 2Dt (2)

where ^(Dx)2amp is the average squareparticle displacement Knowledge ofthe diffusion coefficient gives rise tothe effective hydrodynamic radiusRH of the particles according to theStokesndashEinstein equation

kTD 5 (3)

6phRH

where k is the Boltzmann constantT is the temperature and h is the sol-vent viscosity DLS is a mature tech-nique that has found widespread usein a variety of research settings It isan extremely important tool for de-termining a host of macromolecularproperties such as molecular sizeand shape molecular weight etc

FLUORESCENCECORRELATIONSPECTROSCOPY

Beginning in 1972 Elson Magdeand Webb published a series of pa-pers describing a fluorescence-basedanalogue of DLS which they re-ferred to as fluorescence correlationspectroscopy (FCS)111ndash13 Webb oneof the original participants in theseefforts recently presented a very in-formative history of these develop-ments14 The authors realized that us-ing fluorescence measurements toanalyze the particle motion impartsseveral key advantages over conven-tional DLS Fluorescence is morechemically selective than light scat-tering and allows greater flexibilityin studying the motion of specificanalytes Also fluorescence can bedetected from molecules that aremuch too small to be detected byDLS which allows one to character-ize the motion of small molecules aswell as large macromolecules Final-ly fluorescence analysis opens theway to characterizing chemically re-acting systems Chemical reactionsgenerally do not create a largeenough index of refraction change tobe studied by DLS However spon-taneous chemical reactions can cre-ate fluctuations in the molecular dif-fusion properties andor other fluo-rescence characteristics that couldconceivably be analyzed via fluores-cence detection with much greatersensitivity

Fluorescence correlation spectros-copy was accomplished by focusingan excitation laser beam into the an-alyte solution and then monitoringthe fluorescence generated from thelaser beamrsquos focal region Diffusionof fluorophors into and out of the fo-cal volume altered the local concen-tration of the fluorophors giving riseto spontaneous fluorescence intensityfluctuations that could be analyzed inmuch the same way as the DLS sig-nal By analogy to DLS FCS com-pares the fluorescence intensity mea-sured at time t with the intensitymeasured at a later time t 1 t av-eraged over all values of t This in-formation is contained in an auto-correlation function G(t) A deriva-

APPLIED SPECTROSCOPY 125A

tion of G(t) which assumes a singlecomponent solution and only consid-ers diffusion along the axial dimen-sions of the laser beam yields theequation

1 1G(t) 5 (4)1 2N 1 1 ttd

Note that G(t) decays with t from amaximum at t 5 0 ms The ampli-tude of G(t) depends on the averagenumber of molecules N occupyingthe observation volume (ie the oc-cupancy) and its width depends ontd the average time it takes for amolecule to diffuse through the ob-servation volume The relationshipof td to the molecular diffusion co-efficient is given by

2v0t 5 (5)d 4D

where v0 is the radius of the excita-tion laser beam at its focus Henceas is the case with DLS measuringthe decay rate of G(t) vs t gives thediffusion coefficient of the analyteand from thence its molecular size

Another important aspect of FCSis its ability to characterize chemi-cally reacting systems Spontaneousfluctuations in the local concentra-tion of an analyte can be caused bychemical reactions occurring underequilibrium conditions as well as bymolecular diffusion By characteriz-ing these fluctuations FCS can mea-sure equilibrium constants and reac-tion rate parameters without the needto perturb the system equilibriumElson Magde and Webb derived au-tocorrelation functions for a numberof different scenarios in which thelocal concentration of the analytewas fluctuating due to some type ofspontaneous chemical reaction111

The case that will interest us here iswhere a small ligand molecule bindsto a much larger receptor thus re-ducing the diffusion coefficient ofthe ligand If the ligandndashreceptorcomplex is stable on the time scaleof its transit time through the detec-tion region then the autocorrelationfunction represents a linear combi-nation of two single-component au-tocorrelation functions correspond-ing to the free and bound ligand

2 12N QO i i 1 21 1 tti51 di

G(t) 5 (6)2^Iamp

Here i represents the free or thebound ligand Qi is the molecularbrightness of species i (the averageemission rate of a single molecule ofspecies i) and ^Iamp is the time-aver-aged total signal intensity due to thetotal fluorescence plus the back-ground signal If the diffusion ratesof the different species are sufficient-ly resolved then the autocorrelationfunction can be analyzed to deter-mine the relative concentrations ofbound and unbound ligand provid-ing a measure of the equilibriumbinding constant

In spite of its successful demon-stration and all of its potential ad-vantages FCS was not practical toimplement in those early years DLShad the advantage of being able toanalyze the motion of many uncor-related particles at once through theinterference of multiple scatteredlight waves Since fluorescenceemission is incoherent fluorescencefrom multiple fluorophors does notproduce such effects The only ef-fective way to analyze molecularmotion by fluorescence is to monitorthe individual molecules themselvesThis was simply not possible in the1970s The tricks of the trade for de-tecting single molecules had notbeen invented For one thing the de-tection volume was much too largeOne of the main sources of back-ground noise in single molecule fluo-rescence spectroscopy is scatteringof the excitation laser by the solventSince the backscattered light inten-sity is proportional to the size of thedetection volume the smaller the de-tection volume the better In the earlydays of FCS detection volumes ofpicoliters or greater were employedwhereas modern FCS instrumentsutilize femtoliter-sized detection vol-umes This meant that the backscat-tered light intensity exceeded thefluorescence emission of an individ-ual molecule Another problem wasthat the light collection and detectionefficiencies were far too low to de-

tect the fluorescence emitted by asingle fluorophor One reason for thiswas that the ultrasensitive singlephoton counting detectors used inmodern FCS had yet to be devel-oped In order to detect any signal atall it was necessary to probe 103

to 104 fluorophors at a time resultingin a large average fluorescence sig-nal Long data acquisition times onthe order of tens of minutes to hourswere needed in order to average outthe signal from spurious noise sourc-es and allow the tiny variations inthe average fluorescence signal topeek through In retrospect it is re-ally quite remarkable that the tech-nique worked at all But given itspractical limitations it did not comeinto widespread use for a number ofyears Still the foundation for thedevelopment of FCS and other FFSanalysis methods into a powerful setof modern research tools had beenlaid

SINGLE MOLECULECONFOCAL MICROSCOPY

The crucial advance that revital-ized FCS and led to a suite of alter-native FFS techniques was the com-bination of FCS with single mole-cule confocal microscopy Riglerand co-workers were the first to rec-ognize that confocal microscopywhen used as a spectroscopic anal-ysis tool had great potential forovercoming many of the challengesoriginally encountered with the ear-lier versions of FCS The earliest pa-pers on this subject date back to199215ndash17 Beginning in 1994 Riglerand co-workers1819 and Zare and co-workers2021 demonstrated that con-focal microscopy could be used todirectly detect the fluorescence emit-ted by individual molecules as theydiffused through the microscopic fo-cal volume of the confocal micro-scope This was an important exten-sion of the original single moleculedetection studies reported by Kellerand co-workers7 With this discov-ery the lengthy signal averagingtimes that were needed to measurethe autocorrelation function becamea thing of the past The correlationfunction could be determined based

126A Volume 58 Number 5 2004

focal point

FIG 1 Schematic diagram of a single molecule confocal fluorescence microscope setup used for FFS analysis The inset shows aschematic of the confocal detection volume and a simulated diffusion path of a single molecule through this volume

on a relatively small number of sin-gle molecule fluorescence signalsdetected over a period of a few sec-onds The dramatic rise in the pub-lication rate of FCS related papersthat occurred after these dates atteststo the impact of these important dis-coveries A number of books and re-view articles on single moleculefluorescence detection in solutionand its application to FCS have beenpublished over the years that detailthese advances222ndash29

Confocal microscopy has been animportant biological imaging tool formany years30 Its intended purpose isto create micrometer resolution fluo-rescence images of biological speci-mens and other materials In FFSthe confocal microscope is usedmore as a chemical analysis tool foranalyzing extremely small sub-vol-umes of dilute solutions than as an

imaging device (Fig 1) although itshould be noted that intracellular im-aging is another important areawhere FFS has started making animpact FCS is normally done by fo-cusing an excitation laser beam to itsdiffraction limit using a high numer-ical aperture (NA) microscope objec-tive positioning the focal region intothe analyte solution and monitoringthe fluorescence generated fromwithin the focal volume over timeThe same objective lens also servesto collect fluorescence from the sam-ple an arrangement referred to asepi-illumination A small pinholepositioned at the image plane of theobjective (the position where the im-age comes into focus behind the rearaperture of the objective) acts as aspatial filter to block fluorescencegenerated outside the focal regionfrom reaching the detector thus en-

suring that only the fluorescencegenerated within the focal region canbe detected

The spatial distribution of the lightintensity within the laser beam focusserves as the detection volume Thesize of the detection volume can beestimated by assuming a cylindrical-ly shaped focal volume with radiusv0 and height 2z0 where z0 is theaxial radius of the focal volume v0

and z0 are related to the NA of theobjective the wavelength l of theexcitation light and the index of re-fraction n of the sample mediumaccording to the equations

122l 2nlv 5 z 5 (7)0 0 22middotNA (NA)

In an experiment that utilizes a 13NA objective a 5145-nm laserbeam as the excitation source and an

APPLIED SPECTROSCOPY 127A

aqueous medium (n 5 133) the re-sulting detection volume is 03femtoliters This extremely small de-tection volume is important for sev-eral reasons It suppresses the back-ground noise caused by backscatter-ing of the excitation laser beamthrough Raliegh and Raman scatter-ing processes it enables optical ex-citation of the fluorophors to theirsaturation point using a modest av-erage laser power (1 mW) it en-sures that the number of fluorophorsbeing probed at any given time issmall and it allows samples with ex-tremely small volumes (microlitersor less) to be analyzed

Other aspects of confocal micros-copy that are important for singlemolecule detection include the highcollection efficiency of the objectivelens (25 for a 13 NA oil-im-mersion objective) the high trans-mission efficiency of the opticalcomponents in the wavelength rangeof interest and an efficient singlephoton counting detector Modernsingle photon counting avalanchephotodiode modules are able to de-tect visible photons with 30 to 70quantum efficiency All in all col-lectiondetection efficiencies of 5 to10 are attainable with modern con-focal microscope setups Consider-ing that many fluorophors can emitup to 106 to 108 photons per second(prior to photobleaching) whenpumped near their optical saturationpoint this can lead to photodetectionrates that exceed 105 photons persecond per molecule albeit overbrief time periods

Modern FFS takes advantage ofthe fact that dilute solutions (sub-nanomolar to sub-micromolar) offluorophors exhibit large amplitudefluorescence intensity fluctuationswhen probed by single moleculeconfocal microscopy This allowsthe fluctuations to be characterizedin a matter of seconds rather thanthe tens of minutes to hours neededin the earlier days Large amplitudefluctuations arise because the aver-age number of fluorophors occupy-ing the detection volume (ie theoccupancy) is small compared to thedeviation from the average at any

given time Random diffusion offluorophors into and out of the de-tection volume ensures that the num-ber of fluorophors being probed isnever the same from one moment tothe next Consider for example ananalyte concentration of 1 nM Atthis concentration the average num-ber of fluorophors within a 1-fem-toliter detection volume is 06 mol-ecules This means that on averagethe occupancy fluctuates betweenzero and one corresponding to de-viations from the mean occupancy of06 and 04 respectively If the mi-croscope is properly configured forsingle molecule detection then thefluorescence signal will be charac-terized by lsquolsquoquietrsquorsquo periods duringwhich only background noise is ob-served punctuated by brief intenselsquolsquoburstsrsquorsquo of signal due to the pas-sage of a single molecule throughthe detection volume (see Fig 2a)The durations of the bursts are char-acteristic of the diffusion rate of themolecules with average burst dura-tions typically ranging from a fewtens of microseconds to a few mil-liseconds depending on the mole-culersquos diffusion rate

At fluorophor concentrations be-tween 10 and 100 nM the numberof molecules occupying the detec-tion volume and hence the fluores-cence signal varies about a certainmean value (see Fig 2b) The fluo-rescence data collected under theseconditions is still representative ofindividual molecule transits eventhough more than one molecule isbeing probed at a time The ampli-tude of the autocorrelation functiontaken under these conditions will bereduced due to the inverse relation-ship with the occupancy number (seeFig 2c) As the concentration is in-creased above 100 nM the devia-tion in the occupancy becomes smallcompared to the average fluores-cence signal and the detector startsto reach its saturation point This re-quires lowering the laser power thusreducing the molecular brightness ofthe fluorophors These two effectsplace an upper limit on the fluoro-phor concentration in FCS analysisAt the lower end of the concentra-

tion scale the lengthy time intervalbetween detected molecules be-comes a limiting factor as doesbackground radiation coming fromRaman scattering by the solvent Ingeneral FFS is useful for analyteconcentrations in the range of 01nM to 100 nM It is sometimespossible to attain lower detectionlimits by rapidly scanning the focalvolume of the laser beam relative tothe sample (or vice versa)31 This en-larges the effective detection vol-ume and hence the average molec-ular occupancy without introducingunwanted background radiation

MEASURING THEAUTOCORRELATIONFUNCTION

In confocal microscopy basedFCS single photon counting meth-ods are used to measure the autocor-relation function Experimentallythis is accomplished by accumulat-ing the detected photons into succes-sive time bins of duration Dt Thefluorescence intensity I(t) at anygiven time is equivalent to the num-ber of detected photons ni dividedby the time interval Dt correspond-ing to t 5 iDt The autocorrelationfunction for a given lagtime is cal-culated from Eq 8 after an appro-priately large number of time inter-vals have been accumulated32

2M2k M2k

G(t) 5 (M 2 k) n n nO Oi i1k i1 2i51 i51

(8)

Here M is the total number of timebins and k indicates the time inter-val corresponding to lagtime t 5kDt In practice it is often conve-nient to allow the lengths of the suc-cessive time intervals to vary Pho-tons are initially collected into timebins of a few nanoseconds durationSubsequent photons are then accu-mulated into time bins of increasing-ly longer durations ranging from tensof nanoseconds to seconds Thislsquolsquomultiple-taursquorsquo approach impartssensitivity to fluctuations over abroad range of time scales (nanosec-onds to tens of seconds) without re-quiring excessive data accumula-

128A Volume 58 Number 5 2004

focal point

larr

FIG 2 Time-dependent fluorescence photo-count data and autocorrelation functions ob-tained from static solutions of fluorescent Rho-damine 6G molecules being probed by a sin-gle molecule confocal fluorescence detectionexperiment (a) Fluorescence data (red) ob-tained from a dilute (sub-nM) solution of fluo-rophors Photon bursts from individual mole-cules are clearly resolved The data were re-corded by accumulating the detected photo-counts into successive time bins of 1-msduration (b) Fluorescence data (blue) ob-tained from a more concentrated (10 nM)solution of fluorophors The solution is tooconcentrated for single molecule bursts to beclearly differentiated from the overall fluores-cence (c) Autocorrelation functions typical ofa dilute solution (red) and a more concentrat-ed solution (blue) The solid diamonds are ex-perimental data points and the solid curvesrepresent fits to a modified version of Eq 4that takes into account the lsquolsquotriplet blinkingrsquorsquoeffect at early lagtimes

tions All of these operations can beperformed using a commercial digi-tal correlator available from a num-ber of vendors

APPLICATION OFFLUORESCENCECORRELATIONSPECTROSCOPY IN DRUGDISCOVERY

Conventional diffusional FCS isthe oldest and most widely practicedform of FFS It is an extremely im-portant technique in a large varietyof fields A perusal of the recentbook Fluorescence CorrelationSpectroscopy Theory and Practiceattests to this fact2 A prominent ex-ample of its many uses is its contri-bution to one of the most criticalsteps in the drug discovery pro-cessmdashassessing the binding affinityof the drug candidate for a specifictarget receptor This is done by mon-itoring the change in the diffusiontime of the ligand when it binds toits receptor as illustrated in Fig 3Drug candidates are often small syn-thetic organic molecules but theycan also be peptides or even largebiological macromolecules such asproteins or DNA aptamers One ofthe ways in which they perform theirfunction is by binding to a specificreceptor so as to inhibit its biologicalactivity or to elicit some other bio-

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

APPLIED SPECTROSCOPY 125A

tion of G(t) which assumes a singlecomponent solution and only consid-ers diffusion along the axial dimen-sions of the laser beam yields theequation

1 1G(t) 5 (4)1 2N 1 1 ttd

Note that G(t) decays with t from amaximum at t 5 0 ms The ampli-tude of G(t) depends on the averagenumber of molecules N occupyingthe observation volume (ie the oc-cupancy) and its width depends ontd the average time it takes for amolecule to diffuse through the ob-servation volume The relationshipof td to the molecular diffusion co-efficient is given by

2v0t 5 (5)d 4D

where v0 is the radius of the excita-tion laser beam at its focus Henceas is the case with DLS measuringthe decay rate of G(t) vs t gives thediffusion coefficient of the analyteand from thence its molecular size

Another important aspect of FCSis its ability to characterize chemi-cally reacting systems Spontaneousfluctuations in the local concentra-tion of an analyte can be caused bychemical reactions occurring underequilibrium conditions as well as bymolecular diffusion By characteriz-ing these fluctuations FCS can mea-sure equilibrium constants and reac-tion rate parameters without the needto perturb the system equilibriumElson Magde and Webb derived au-tocorrelation functions for a numberof different scenarios in which thelocal concentration of the analytewas fluctuating due to some type ofspontaneous chemical reaction111

The case that will interest us here iswhere a small ligand molecule bindsto a much larger receptor thus re-ducing the diffusion coefficient ofthe ligand If the ligandndashreceptorcomplex is stable on the time scaleof its transit time through the detec-tion region then the autocorrelationfunction represents a linear combi-nation of two single-component au-tocorrelation functions correspond-ing to the free and bound ligand

2 12N QO i i 1 21 1 tti51 di

G(t) 5 (6)2^Iamp

Here i represents the free or thebound ligand Qi is the molecularbrightness of species i (the averageemission rate of a single molecule ofspecies i) and ^Iamp is the time-aver-aged total signal intensity due to thetotal fluorescence plus the back-ground signal If the diffusion ratesof the different species are sufficient-ly resolved then the autocorrelationfunction can be analyzed to deter-mine the relative concentrations ofbound and unbound ligand provid-ing a measure of the equilibriumbinding constant

In spite of its successful demon-stration and all of its potential ad-vantages FCS was not practical toimplement in those early years DLShad the advantage of being able toanalyze the motion of many uncor-related particles at once through theinterference of multiple scatteredlight waves Since fluorescenceemission is incoherent fluorescencefrom multiple fluorophors does notproduce such effects The only ef-fective way to analyze molecularmotion by fluorescence is to monitorthe individual molecules themselvesThis was simply not possible in the1970s The tricks of the trade for de-tecting single molecules had notbeen invented For one thing the de-tection volume was much too largeOne of the main sources of back-ground noise in single molecule fluo-rescence spectroscopy is scatteringof the excitation laser by the solventSince the backscattered light inten-sity is proportional to the size of thedetection volume the smaller the de-tection volume the better In the earlydays of FCS detection volumes ofpicoliters or greater were employedwhereas modern FCS instrumentsutilize femtoliter-sized detection vol-umes This meant that the backscat-tered light intensity exceeded thefluorescence emission of an individ-ual molecule Another problem wasthat the light collection and detectionefficiencies were far too low to de-

tect the fluorescence emitted by asingle fluorophor One reason for thiswas that the ultrasensitive singlephoton counting detectors used inmodern FCS had yet to be devel-oped In order to detect any signal atall it was necessary to probe 103

to 104 fluorophors at a time resultingin a large average fluorescence sig-nal Long data acquisition times onthe order of tens of minutes to hourswere needed in order to average outthe signal from spurious noise sourc-es and allow the tiny variations inthe average fluorescence signal topeek through In retrospect it is re-ally quite remarkable that the tech-nique worked at all But given itspractical limitations it did not comeinto widespread use for a number ofyears Still the foundation for thedevelopment of FCS and other FFSanalysis methods into a powerful setof modern research tools had beenlaid

SINGLE MOLECULECONFOCAL MICROSCOPY

The crucial advance that revital-ized FCS and led to a suite of alter-native FFS techniques was the com-bination of FCS with single mole-cule confocal microscopy Riglerand co-workers were the first to rec-ognize that confocal microscopywhen used as a spectroscopic anal-ysis tool had great potential forovercoming many of the challengesoriginally encountered with the ear-lier versions of FCS The earliest pa-pers on this subject date back to199215ndash17 Beginning in 1994 Riglerand co-workers1819 and Zare and co-workers2021 demonstrated that con-focal microscopy could be used todirectly detect the fluorescence emit-ted by individual molecules as theydiffused through the microscopic fo-cal volume of the confocal micro-scope This was an important exten-sion of the original single moleculedetection studies reported by Kellerand co-workers7 With this discov-ery the lengthy signal averagingtimes that were needed to measurethe autocorrelation function becamea thing of the past The correlationfunction could be determined based

126A Volume 58 Number 5 2004

focal point

FIG 1 Schematic diagram of a single molecule confocal fluorescence microscope setup used for FFS analysis The inset shows aschematic of the confocal detection volume and a simulated diffusion path of a single molecule through this volume

on a relatively small number of sin-gle molecule fluorescence signalsdetected over a period of a few sec-onds The dramatic rise in the pub-lication rate of FCS related papersthat occurred after these dates atteststo the impact of these important dis-coveries A number of books and re-view articles on single moleculefluorescence detection in solutionand its application to FCS have beenpublished over the years that detailthese advances222ndash29

Confocal microscopy has been animportant biological imaging tool formany years30 Its intended purpose isto create micrometer resolution fluo-rescence images of biological speci-mens and other materials In FFSthe confocal microscope is usedmore as a chemical analysis tool foranalyzing extremely small sub-vol-umes of dilute solutions than as an

imaging device (Fig 1) although itshould be noted that intracellular im-aging is another important areawhere FFS has started making animpact FCS is normally done by fo-cusing an excitation laser beam to itsdiffraction limit using a high numer-ical aperture (NA) microscope objec-tive positioning the focal region intothe analyte solution and monitoringthe fluorescence generated fromwithin the focal volume over timeThe same objective lens also servesto collect fluorescence from the sam-ple an arrangement referred to asepi-illumination A small pinholepositioned at the image plane of theobjective (the position where the im-age comes into focus behind the rearaperture of the objective) acts as aspatial filter to block fluorescencegenerated outside the focal regionfrom reaching the detector thus en-

suring that only the fluorescencegenerated within the focal region canbe detected

The spatial distribution of the lightintensity within the laser beam focusserves as the detection volume Thesize of the detection volume can beestimated by assuming a cylindrical-ly shaped focal volume with radiusv0 and height 2z0 where z0 is theaxial radius of the focal volume v0

and z0 are related to the NA of theobjective the wavelength l of theexcitation light and the index of re-fraction n of the sample mediumaccording to the equations

122l 2nlv 5 z 5 (7)0 0 22middotNA (NA)

In an experiment that utilizes a 13NA objective a 5145-nm laserbeam as the excitation source and an

APPLIED SPECTROSCOPY 127A

aqueous medium (n 5 133) the re-sulting detection volume is 03femtoliters This extremely small de-tection volume is important for sev-eral reasons It suppresses the back-ground noise caused by backscatter-ing of the excitation laser beamthrough Raliegh and Raman scatter-ing processes it enables optical ex-citation of the fluorophors to theirsaturation point using a modest av-erage laser power (1 mW) it en-sures that the number of fluorophorsbeing probed at any given time issmall and it allows samples with ex-tremely small volumes (microlitersor less) to be analyzed

Other aspects of confocal micros-copy that are important for singlemolecule detection include the highcollection efficiency of the objectivelens (25 for a 13 NA oil-im-mersion objective) the high trans-mission efficiency of the opticalcomponents in the wavelength rangeof interest and an efficient singlephoton counting detector Modernsingle photon counting avalanchephotodiode modules are able to de-tect visible photons with 30 to 70quantum efficiency All in all col-lectiondetection efficiencies of 5 to10 are attainable with modern con-focal microscope setups Consider-ing that many fluorophors can emitup to 106 to 108 photons per second(prior to photobleaching) whenpumped near their optical saturationpoint this can lead to photodetectionrates that exceed 105 photons persecond per molecule albeit overbrief time periods

Modern FFS takes advantage ofthe fact that dilute solutions (sub-nanomolar to sub-micromolar) offluorophors exhibit large amplitudefluorescence intensity fluctuationswhen probed by single moleculeconfocal microscopy This allowsthe fluctuations to be characterizedin a matter of seconds rather thanthe tens of minutes to hours neededin the earlier days Large amplitudefluctuations arise because the aver-age number of fluorophors occupy-ing the detection volume (ie theoccupancy) is small compared to thedeviation from the average at any

given time Random diffusion offluorophors into and out of the de-tection volume ensures that the num-ber of fluorophors being probed isnever the same from one moment tothe next Consider for example ananalyte concentration of 1 nM Atthis concentration the average num-ber of fluorophors within a 1-fem-toliter detection volume is 06 mol-ecules This means that on averagethe occupancy fluctuates betweenzero and one corresponding to de-viations from the mean occupancy of06 and 04 respectively If the mi-croscope is properly configured forsingle molecule detection then thefluorescence signal will be charac-terized by lsquolsquoquietrsquorsquo periods duringwhich only background noise is ob-served punctuated by brief intenselsquolsquoburstsrsquorsquo of signal due to the pas-sage of a single molecule throughthe detection volume (see Fig 2a)The durations of the bursts are char-acteristic of the diffusion rate of themolecules with average burst dura-tions typically ranging from a fewtens of microseconds to a few mil-liseconds depending on the mole-culersquos diffusion rate

At fluorophor concentrations be-tween 10 and 100 nM the numberof molecules occupying the detec-tion volume and hence the fluores-cence signal varies about a certainmean value (see Fig 2b) The fluo-rescence data collected under theseconditions is still representative ofindividual molecule transits eventhough more than one molecule isbeing probed at a time The ampli-tude of the autocorrelation functiontaken under these conditions will bereduced due to the inverse relation-ship with the occupancy number (seeFig 2c) As the concentration is in-creased above 100 nM the devia-tion in the occupancy becomes smallcompared to the average fluores-cence signal and the detector startsto reach its saturation point This re-quires lowering the laser power thusreducing the molecular brightness ofthe fluorophors These two effectsplace an upper limit on the fluoro-phor concentration in FCS analysisAt the lower end of the concentra-

tion scale the lengthy time intervalbetween detected molecules be-comes a limiting factor as doesbackground radiation coming fromRaman scattering by the solvent Ingeneral FFS is useful for analyteconcentrations in the range of 01nM to 100 nM It is sometimespossible to attain lower detectionlimits by rapidly scanning the focalvolume of the laser beam relative tothe sample (or vice versa)31 This en-larges the effective detection vol-ume and hence the average molec-ular occupancy without introducingunwanted background radiation

MEASURING THEAUTOCORRELATIONFUNCTION

In confocal microscopy basedFCS single photon counting meth-ods are used to measure the autocor-relation function Experimentallythis is accomplished by accumulat-ing the detected photons into succes-sive time bins of duration Dt Thefluorescence intensity I(t) at anygiven time is equivalent to the num-ber of detected photons ni dividedby the time interval Dt correspond-ing to t 5 iDt The autocorrelationfunction for a given lagtime is cal-culated from Eq 8 after an appro-priately large number of time inter-vals have been accumulated32

2M2k M2k

G(t) 5 (M 2 k) n n nO Oi i1k i1 2i51 i51

(8)

Here M is the total number of timebins and k indicates the time inter-val corresponding to lagtime t 5kDt In practice it is often conve-nient to allow the lengths of the suc-cessive time intervals to vary Pho-tons are initially collected into timebins of a few nanoseconds durationSubsequent photons are then accu-mulated into time bins of increasing-ly longer durations ranging from tensof nanoseconds to seconds Thislsquolsquomultiple-taursquorsquo approach impartssensitivity to fluctuations over abroad range of time scales (nanosec-onds to tens of seconds) without re-quiring excessive data accumula-

128A Volume 58 Number 5 2004

focal point

larr

FIG 2 Time-dependent fluorescence photo-count data and autocorrelation functions ob-tained from static solutions of fluorescent Rho-damine 6G molecules being probed by a sin-gle molecule confocal fluorescence detectionexperiment (a) Fluorescence data (red) ob-tained from a dilute (sub-nM) solution of fluo-rophors Photon bursts from individual mole-cules are clearly resolved The data were re-corded by accumulating the detected photo-counts into successive time bins of 1-msduration (b) Fluorescence data (blue) ob-tained from a more concentrated (10 nM)solution of fluorophors The solution is tooconcentrated for single molecule bursts to beclearly differentiated from the overall fluores-cence (c) Autocorrelation functions typical ofa dilute solution (red) and a more concentrat-ed solution (blue) The solid diamonds are ex-perimental data points and the solid curvesrepresent fits to a modified version of Eq 4that takes into account the lsquolsquotriplet blinkingrsquorsquoeffect at early lagtimes

tions All of these operations can beperformed using a commercial digi-tal correlator available from a num-ber of vendors

APPLICATION OFFLUORESCENCECORRELATIONSPECTROSCOPY IN DRUGDISCOVERY

Conventional diffusional FCS isthe oldest and most widely practicedform of FFS It is an extremely im-portant technique in a large varietyof fields A perusal of the recentbook Fluorescence CorrelationSpectroscopy Theory and Practiceattests to this fact2 A prominent ex-ample of its many uses is its contri-bution to one of the most criticalsteps in the drug discovery pro-cessmdashassessing the binding affinityof the drug candidate for a specifictarget receptor This is done by mon-itoring the change in the diffusiontime of the ligand when it binds toits receptor as illustrated in Fig 3Drug candidates are often small syn-thetic organic molecules but theycan also be peptides or even largebiological macromolecules such asproteins or DNA aptamers One ofthe ways in which they perform theirfunction is by binding to a specificreceptor so as to inhibit its biologicalactivity or to elicit some other bio-

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

126A Volume 58 Number 5 2004

focal point

FIG 1 Schematic diagram of a single molecule confocal fluorescence microscope setup used for FFS analysis The inset shows aschematic of the confocal detection volume and a simulated diffusion path of a single molecule through this volume

on a relatively small number of sin-gle molecule fluorescence signalsdetected over a period of a few sec-onds The dramatic rise in the pub-lication rate of FCS related papersthat occurred after these dates atteststo the impact of these important dis-coveries A number of books and re-view articles on single moleculefluorescence detection in solutionand its application to FCS have beenpublished over the years that detailthese advances222ndash29

Confocal microscopy has been animportant biological imaging tool formany years30 Its intended purpose isto create micrometer resolution fluo-rescence images of biological speci-mens and other materials In FFSthe confocal microscope is usedmore as a chemical analysis tool foranalyzing extremely small sub-vol-umes of dilute solutions than as an

imaging device (Fig 1) although itshould be noted that intracellular im-aging is another important areawhere FFS has started making animpact FCS is normally done by fo-cusing an excitation laser beam to itsdiffraction limit using a high numer-ical aperture (NA) microscope objec-tive positioning the focal region intothe analyte solution and monitoringthe fluorescence generated fromwithin the focal volume over timeThe same objective lens also servesto collect fluorescence from the sam-ple an arrangement referred to asepi-illumination A small pinholepositioned at the image plane of theobjective (the position where the im-age comes into focus behind the rearaperture of the objective) acts as aspatial filter to block fluorescencegenerated outside the focal regionfrom reaching the detector thus en-

suring that only the fluorescencegenerated within the focal region canbe detected

The spatial distribution of the lightintensity within the laser beam focusserves as the detection volume Thesize of the detection volume can beestimated by assuming a cylindrical-ly shaped focal volume with radiusv0 and height 2z0 where z0 is theaxial radius of the focal volume v0

and z0 are related to the NA of theobjective the wavelength l of theexcitation light and the index of re-fraction n of the sample mediumaccording to the equations

122l 2nlv 5 z 5 (7)0 0 22middotNA (NA)

In an experiment that utilizes a 13NA objective a 5145-nm laserbeam as the excitation source and an

APPLIED SPECTROSCOPY 127A

aqueous medium (n 5 133) the re-sulting detection volume is 03femtoliters This extremely small de-tection volume is important for sev-eral reasons It suppresses the back-ground noise caused by backscatter-ing of the excitation laser beamthrough Raliegh and Raman scatter-ing processes it enables optical ex-citation of the fluorophors to theirsaturation point using a modest av-erage laser power (1 mW) it en-sures that the number of fluorophorsbeing probed at any given time issmall and it allows samples with ex-tremely small volumes (microlitersor less) to be analyzed

Other aspects of confocal micros-copy that are important for singlemolecule detection include the highcollection efficiency of the objectivelens (25 for a 13 NA oil-im-mersion objective) the high trans-mission efficiency of the opticalcomponents in the wavelength rangeof interest and an efficient singlephoton counting detector Modernsingle photon counting avalanchephotodiode modules are able to de-tect visible photons with 30 to 70quantum efficiency All in all col-lectiondetection efficiencies of 5 to10 are attainable with modern con-focal microscope setups Consider-ing that many fluorophors can emitup to 106 to 108 photons per second(prior to photobleaching) whenpumped near their optical saturationpoint this can lead to photodetectionrates that exceed 105 photons persecond per molecule albeit overbrief time periods

Modern FFS takes advantage ofthe fact that dilute solutions (sub-nanomolar to sub-micromolar) offluorophors exhibit large amplitudefluorescence intensity fluctuationswhen probed by single moleculeconfocal microscopy This allowsthe fluctuations to be characterizedin a matter of seconds rather thanthe tens of minutes to hours neededin the earlier days Large amplitudefluctuations arise because the aver-age number of fluorophors occupy-ing the detection volume (ie theoccupancy) is small compared to thedeviation from the average at any

given time Random diffusion offluorophors into and out of the de-tection volume ensures that the num-ber of fluorophors being probed isnever the same from one moment tothe next Consider for example ananalyte concentration of 1 nM Atthis concentration the average num-ber of fluorophors within a 1-fem-toliter detection volume is 06 mol-ecules This means that on averagethe occupancy fluctuates betweenzero and one corresponding to de-viations from the mean occupancy of06 and 04 respectively If the mi-croscope is properly configured forsingle molecule detection then thefluorescence signal will be charac-terized by lsquolsquoquietrsquorsquo periods duringwhich only background noise is ob-served punctuated by brief intenselsquolsquoburstsrsquorsquo of signal due to the pas-sage of a single molecule throughthe detection volume (see Fig 2a)The durations of the bursts are char-acteristic of the diffusion rate of themolecules with average burst dura-tions typically ranging from a fewtens of microseconds to a few mil-liseconds depending on the mole-culersquos diffusion rate

At fluorophor concentrations be-tween 10 and 100 nM the numberof molecules occupying the detec-tion volume and hence the fluores-cence signal varies about a certainmean value (see Fig 2b) The fluo-rescence data collected under theseconditions is still representative ofindividual molecule transits eventhough more than one molecule isbeing probed at a time The ampli-tude of the autocorrelation functiontaken under these conditions will bereduced due to the inverse relation-ship with the occupancy number (seeFig 2c) As the concentration is in-creased above 100 nM the devia-tion in the occupancy becomes smallcompared to the average fluores-cence signal and the detector startsto reach its saturation point This re-quires lowering the laser power thusreducing the molecular brightness ofthe fluorophors These two effectsplace an upper limit on the fluoro-phor concentration in FCS analysisAt the lower end of the concentra-

tion scale the lengthy time intervalbetween detected molecules be-comes a limiting factor as doesbackground radiation coming fromRaman scattering by the solvent Ingeneral FFS is useful for analyteconcentrations in the range of 01nM to 100 nM It is sometimespossible to attain lower detectionlimits by rapidly scanning the focalvolume of the laser beam relative tothe sample (or vice versa)31 This en-larges the effective detection vol-ume and hence the average molec-ular occupancy without introducingunwanted background radiation

MEASURING THEAUTOCORRELATIONFUNCTION

In confocal microscopy basedFCS single photon counting meth-ods are used to measure the autocor-relation function Experimentallythis is accomplished by accumulat-ing the detected photons into succes-sive time bins of duration Dt Thefluorescence intensity I(t) at anygiven time is equivalent to the num-ber of detected photons ni dividedby the time interval Dt correspond-ing to t 5 iDt The autocorrelationfunction for a given lagtime is cal-culated from Eq 8 after an appro-priately large number of time inter-vals have been accumulated32

2M2k M2k

G(t) 5 (M 2 k) n n nO Oi i1k i1 2i51 i51

(8)

Here M is the total number of timebins and k indicates the time inter-val corresponding to lagtime t 5kDt In practice it is often conve-nient to allow the lengths of the suc-cessive time intervals to vary Pho-tons are initially collected into timebins of a few nanoseconds durationSubsequent photons are then accu-mulated into time bins of increasing-ly longer durations ranging from tensof nanoseconds to seconds Thislsquolsquomultiple-taursquorsquo approach impartssensitivity to fluctuations over abroad range of time scales (nanosec-onds to tens of seconds) without re-quiring excessive data accumula-

128A Volume 58 Number 5 2004

focal point

larr

FIG 2 Time-dependent fluorescence photo-count data and autocorrelation functions ob-tained from static solutions of fluorescent Rho-damine 6G molecules being probed by a sin-gle molecule confocal fluorescence detectionexperiment (a) Fluorescence data (red) ob-tained from a dilute (sub-nM) solution of fluo-rophors Photon bursts from individual mole-cules are clearly resolved The data were re-corded by accumulating the detected photo-counts into successive time bins of 1-msduration (b) Fluorescence data (blue) ob-tained from a more concentrated (10 nM)solution of fluorophors The solution is tooconcentrated for single molecule bursts to beclearly differentiated from the overall fluores-cence (c) Autocorrelation functions typical ofa dilute solution (red) and a more concentrat-ed solution (blue) The solid diamonds are ex-perimental data points and the solid curvesrepresent fits to a modified version of Eq 4that takes into account the lsquolsquotriplet blinkingrsquorsquoeffect at early lagtimes

tions All of these operations can beperformed using a commercial digi-tal correlator available from a num-ber of vendors

APPLICATION OFFLUORESCENCECORRELATIONSPECTROSCOPY IN DRUGDISCOVERY

Conventional diffusional FCS isthe oldest and most widely practicedform of FFS It is an extremely im-portant technique in a large varietyof fields A perusal of the recentbook Fluorescence CorrelationSpectroscopy Theory and Practiceattests to this fact2 A prominent ex-ample of its many uses is its contri-bution to one of the most criticalsteps in the drug discovery pro-cessmdashassessing the binding affinityof the drug candidate for a specifictarget receptor This is done by mon-itoring the change in the diffusiontime of the ligand when it binds toits receptor as illustrated in Fig 3Drug candidates are often small syn-thetic organic molecules but theycan also be peptides or even largebiological macromolecules such asproteins or DNA aptamers One ofthe ways in which they perform theirfunction is by binding to a specificreceptor so as to inhibit its biologicalactivity or to elicit some other bio-

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

APPLIED SPECTROSCOPY 127A

aqueous medium (n 5 133) the re-sulting detection volume is 03femtoliters This extremely small de-tection volume is important for sev-eral reasons It suppresses the back-ground noise caused by backscatter-ing of the excitation laser beamthrough Raliegh and Raman scatter-ing processes it enables optical ex-citation of the fluorophors to theirsaturation point using a modest av-erage laser power (1 mW) it en-sures that the number of fluorophorsbeing probed at any given time issmall and it allows samples with ex-tremely small volumes (microlitersor less) to be analyzed

Other aspects of confocal micros-copy that are important for singlemolecule detection include the highcollection efficiency of the objectivelens (25 for a 13 NA oil-im-mersion objective) the high trans-mission efficiency of the opticalcomponents in the wavelength rangeof interest and an efficient singlephoton counting detector Modernsingle photon counting avalanchephotodiode modules are able to de-tect visible photons with 30 to 70quantum efficiency All in all col-lectiondetection efficiencies of 5 to10 are attainable with modern con-focal microscope setups Consider-ing that many fluorophors can emitup to 106 to 108 photons per second(prior to photobleaching) whenpumped near their optical saturationpoint this can lead to photodetectionrates that exceed 105 photons persecond per molecule albeit overbrief time periods

Modern FFS takes advantage ofthe fact that dilute solutions (sub-nanomolar to sub-micromolar) offluorophors exhibit large amplitudefluorescence intensity fluctuationswhen probed by single moleculeconfocal microscopy This allowsthe fluctuations to be characterizedin a matter of seconds rather thanthe tens of minutes to hours neededin the earlier days Large amplitudefluctuations arise because the aver-age number of fluorophors occupy-ing the detection volume (ie theoccupancy) is small compared to thedeviation from the average at any

given time Random diffusion offluorophors into and out of the de-tection volume ensures that the num-ber of fluorophors being probed isnever the same from one moment tothe next Consider for example ananalyte concentration of 1 nM Atthis concentration the average num-ber of fluorophors within a 1-fem-toliter detection volume is 06 mol-ecules This means that on averagethe occupancy fluctuates betweenzero and one corresponding to de-viations from the mean occupancy of06 and 04 respectively If the mi-croscope is properly configured forsingle molecule detection then thefluorescence signal will be charac-terized by lsquolsquoquietrsquorsquo periods duringwhich only background noise is ob-served punctuated by brief intenselsquolsquoburstsrsquorsquo of signal due to the pas-sage of a single molecule throughthe detection volume (see Fig 2a)The durations of the bursts are char-acteristic of the diffusion rate of themolecules with average burst dura-tions typically ranging from a fewtens of microseconds to a few mil-liseconds depending on the mole-culersquos diffusion rate

At fluorophor concentrations be-tween 10 and 100 nM the numberof molecules occupying the detec-tion volume and hence the fluores-cence signal varies about a certainmean value (see Fig 2b) The fluo-rescence data collected under theseconditions is still representative ofindividual molecule transits eventhough more than one molecule isbeing probed at a time The ampli-tude of the autocorrelation functiontaken under these conditions will bereduced due to the inverse relation-ship with the occupancy number (seeFig 2c) As the concentration is in-creased above 100 nM the devia-tion in the occupancy becomes smallcompared to the average fluores-cence signal and the detector startsto reach its saturation point This re-quires lowering the laser power thusreducing the molecular brightness ofthe fluorophors These two effectsplace an upper limit on the fluoro-phor concentration in FCS analysisAt the lower end of the concentra-

tion scale the lengthy time intervalbetween detected molecules be-comes a limiting factor as doesbackground radiation coming fromRaman scattering by the solvent Ingeneral FFS is useful for analyteconcentrations in the range of 01nM to 100 nM It is sometimespossible to attain lower detectionlimits by rapidly scanning the focalvolume of the laser beam relative tothe sample (or vice versa)31 This en-larges the effective detection vol-ume and hence the average molec-ular occupancy without introducingunwanted background radiation

MEASURING THEAUTOCORRELATIONFUNCTION

In confocal microscopy basedFCS single photon counting meth-ods are used to measure the autocor-relation function Experimentallythis is accomplished by accumulat-ing the detected photons into succes-sive time bins of duration Dt Thefluorescence intensity I(t) at anygiven time is equivalent to the num-ber of detected photons ni dividedby the time interval Dt correspond-ing to t 5 iDt The autocorrelationfunction for a given lagtime is cal-culated from Eq 8 after an appro-priately large number of time inter-vals have been accumulated32

2M2k M2k

G(t) 5 (M 2 k) n n nO Oi i1k i1 2i51 i51

(8)

Here M is the total number of timebins and k indicates the time inter-val corresponding to lagtime t 5kDt In practice it is often conve-nient to allow the lengths of the suc-cessive time intervals to vary Pho-tons are initially collected into timebins of a few nanoseconds durationSubsequent photons are then accu-mulated into time bins of increasing-ly longer durations ranging from tensof nanoseconds to seconds Thislsquolsquomultiple-taursquorsquo approach impartssensitivity to fluctuations over abroad range of time scales (nanosec-onds to tens of seconds) without re-quiring excessive data accumula-

128A Volume 58 Number 5 2004

focal point

larr

FIG 2 Time-dependent fluorescence photo-count data and autocorrelation functions ob-tained from static solutions of fluorescent Rho-damine 6G molecules being probed by a sin-gle molecule confocal fluorescence detectionexperiment (a) Fluorescence data (red) ob-tained from a dilute (sub-nM) solution of fluo-rophors Photon bursts from individual mole-cules are clearly resolved The data were re-corded by accumulating the detected photo-counts into successive time bins of 1-msduration (b) Fluorescence data (blue) ob-tained from a more concentrated (10 nM)solution of fluorophors The solution is tooconcentrated for single molecule bursts to beclearly differentiated from the overall fluores-cence (c) Autocorrelation functions typical ofa dilute solution (red) and a more concentrat-ed solution (blue) The solid diamonds are ex-perimental data points and the solid curvesrepresent fits to a modified version of Eq 4that takes into account the lsquolsquotriplet blinkingrsquorsquoeffect at early lagtimes

tions All of these operations can beperformed using a commercial digi-tal correlator available from a num-ber of vendors

APPLICATION OFFLUORESCENCECORRELATIONSPECTROSCOPY IN DRUGDISCOVERY

Conventional diffusional FCS isthe oldest and most widely practicedform of FFS It is an extremely im-portant technique in a large varietyof fields A perusal of the recentbook Fluorescence CorrelationSpectroscopy Theory and Practiceattests to this fact2 A prominent ex-ample of its many uses is its contri-bution to one of the most criticalsteps in the drug discovery pro-cessmdashassessing the binding affinityof the drug candidate for a specifictarget receptor This is done by mon-itoring the change in the diffusiontime of the ligand when it binds toits receptor as illustrated in Fig 3Drug candidates are often small syn-thetic organic molecules but theycan also be peptides or even largebiological macromolecules such asproteins or DNA aptamers One ofthe ways in which they perform theirfunction is by binding to a specificreceptor so as to inhibit its biologicalactivity or to elicit some other bio-

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

128A Volume 58 Number 5 2004

focal point

larr

FIG 2 Time-dependent fluorescence photo-count data and autocorrelation functions ob-tained from static solutions of fluorescent Rho-damine 6G molecules being probed by a sin-gle molecule confocal fluorescence detectionexperiment (a) Fluorescence data (red) ob-tained from a dilute (sub-nM) solution of fluo-rophors Photon bursts from individual mole-cules are clearly resolved The data were re-corded by accumulating the detected photo-counts into successive time bins of 1-msduration (b) Fluorescence data (blue) ob-tained from a more concentrated (10 nM)solution of fluorophors The solution is tooconcentrated for single molecule bursts to beclearly differentiated from the overall fluores-cence (c) Autocorrelation functions typical ofa dilute solution (red) and a more concentrat-ed solution (blue) The solid diamonds are ex-perimental data points and the solid curvesrepresent fits to a modified version of Eq 4that takes into account the lsquolsquotriplet blinkingrsquorsquoeffect at early lagtimes

tions All of these operations can beperformed using a commercial digi-tal correlator available from a num-ber of vendors

APPLICATION OFFLUORESCENCECORRELATIONSPECTROSCOPY IN DRUGDISCOVERY

Conventional diffusional FCS isthe oldest and most widely practicedform of FFS It is an extremely im-portant technique in a large varietyof fields A perusal of the recentbook Fluorescence CorrelationSpectroscopy Theory and Practiceattests to this fact2 A prominent ex-ample of its many uses is its contri-bution to one of the most criticalsteps in the drug discovery pro-cessmdashassessing the binding affinityof the drug candidate for a specifictarget receptor This is done by mon-itoring the change in the diffusiontime of the ligand when it binds toits receptor as illustrated in Fig 3Drug candidates are often small syn-thetic organic molecules but theycan also be peptides or even largebiological macromolecules such asproteins or DNA aptamers One ofthe ways in which they perform theirfunction is by binding to a specificreceptor so as to inhibit its biologicalactivity or to elicit some other bio-

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

APPLIED SPECTROSCOPY 129A

FIG 3 Diffusional FCS for characterizing a ligandndashreceptor binding interaction As a small fluorescently labeled ligand binds to alarger receptor the translational diffusion rate of the ligand decreases resulting in a shift in the width of the correlation function tolonger lagtimes Each correlation function can be analyzed to determine the concentrations of bound and unbound ligands Typicalacquisition times for each correlation function are seconds to tens of seconds When the reaction occurs on a slower time scale thanthe acquisition time the width of the correlation function can be monitored to follow the progress of the reaction over time Forfaster reactions the correlation function can be measured for different receptor concentrations to determine the binding affinity(Copyright Karl Zeiss Jena GmbH)

logical response Target receptorscan be proteins such as enzymes orantibodies specific sequences ofDNA or RNA or cell surface recep-tors

Fluorescence correlation spectros-copy is being used in drug discoveryresearch in two distinct ways bothof which take advantage of the abil-ity to carry out ligandndashreceptor bind-ing assays by resolving the transla-tional diffusion rates of the boundand unbound ligands Firstly FCShas become an important comple-mentary technique for the detailedbiophysical investigation of specificligandndashreceptor complexes Once apotential drug candidate has beenidentified either by high-throughputscreening or rational drug design abattery of experiments need to bedone to assess the binding affinityand kinetic rate parameters for the

biomolecular interactions involvedThis phase of the process is referredto as secondary screening Fluores-cence-based methods are widelyused because of their high sensitivityand their ability to operate in a ho-mogenous assay format (ie theability to perform the assay in situwithout separating bound from un-bound ligands) One of the mostpopular fluorescence methods beingused for this purpose is fluorescencepolarization (FP) analysis33 In con-trast to FCS which measures trans-lational diffusion FP measures thechange in rotational diffusion ratethat takes place when a fluorescentlylabeled ligand binds to a larger re-ceptor FP has one key advantageover FCS in its ability to resolve thebound and unbound fractions of theligandndashreceptor complex Thiscomes from the fact that the rota-

tional diffusion rate as monitored byFP changes as a function of 1R 3

H

whereas the translational diffusionrate which is monitored by FCSchanges as 1RH This gives FPgreater sensitivity to small changesin the molecular size as compared toFCS However FCS has other char-acteristics that make it a good com-plement to FP in secondary screen-ing applications As noted by Matay-oshi and Swift FP is only effectivewhen the rotational diffusion rate isno more than five to ten times largerthan the fluorescence emission rateof the fluorophor34 This makes larg-er complexes with slow rotationaldiffusion rates difficult to analyzeusing FP Yet this is precisely whereFCS is at its best in its ability tomonitor the diffusion of large slow-ly diffusing molecular complexes

One area where this advantage

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

130A Volume 58 Number 5 2004

focal point

clearly comes into play is in theanalysis of molecular aggregation aprominent example of which is theformation of amyloid b-peptide (Ab)fibrils Ab is produced in humansthrough abnormalities in the enzy-matic digestion of a protein knownas amyloid precursor protein Onceformed these peptides can self-as-semble into thin cylindricallyshaped macromolecular complexestypically measuring between fiveand ten nanometers in width andtens to hundreds of nanometers inlength These complexes are knownas fibrils and they can interact witheach other even further to formplaque residues that deposit into re-gions of the brain and central ner-vous system Such processes aresymptomatic of Alzheimerrsquos diseaseand other neurological disorders Li-gands are being sought that can ei-ther inhibit the assembly of the fi-brils or disrupt them once formedRigler and co-workers have demon-strated that FCS is extremely effec-tive in monitoring the formation ofAb fibrils in vitro35 They accom-plished this feat by monitoring theautocorrelation function for solutionscontaining fluorescently labeled Abmonomers under conditions whereaggregation of the monomers occursover a time period of tens of minutesto hours Because of the large sizedifference between the peptidemonomers and the fibrils fibril for-mation could be readily observedand quantified based on the analysisof the autocorrelation function Theauthors were also able to show thatFCS could be used to quantify theability of various Ab ligands to sup-press fibril formation Hence FCSwas shown to be a very importanttool for characterizing the effects ofdrugs against the types of disordersthat involve formation of large mo-lecular aggregates

Another way in which FCS out-performs conventional fluorescencetechniques like FP is in the secondtype of drug discovery applicationmdashhigh-throughput screening of drugcandidate libraries3ndash6 High-through-put screening also referred to as pri-mary screening is the process by

which one analyzes a library con-sisting of hundreds or even thou-sands of individual compounds inorder to identify those compoundsthat possess a desired biological ac-tivity The samples are typically ar-rayed out in individual sample wellson a spatially addressable microtiterplate and an independent assay isperformed on each sample to assessits biological activity Several factorshave conspired to require screeningof ever larger numbers of com-pounds using smaller sample vol-umes and in shorter time The num-ber of receptors that can potentiallybe targeted by drugs continues to in-crease at a dramatic pace thanks inlarge part to the success of the hu-man genome project and the accel-erated pace of proteomics researchIf there are no known ligands for agiven receptor or if the receptorrsquosmolecular structure is unknownthere is little choice but to screen asmany compounds as possible in thehope that some compound willemerge with sufficient binding affin-ity to serve as a lead for the devel-opment of a new drug This is adaunting task given that many phar-maceutical companies possess librar-ies of tens to hundreds of thousandsof compounds any one of whichcould be a crucial new lead To im-prove the screening efficiency andminimize reagent costs it has be-come important to maximize thenumber of compounds per arraywhile minimizing the individualsample volumes Microtiter platescontaining as many as 2080 samplewells each with individual samplevolumes of 1 mL or less have beendeveloped for this purpose (Fig 4)Thanks in large measure to their ul-tra-high sensitivity fluorescence-based detection methods are current-ly the most popular choice for per-forming these highly miniaturizedassays

Fluorescence polarization is anexample of a macroscopic fluores-cence method These methods col-lect fluorescence from a large en-semble of molecules integrated overthe entire volume of the sampleThey are extremely effective when

used in the standard 96-well micro-titer plate format where the 100 mLsample volume generates plenty offluorescence signal even at sub-nanomolar analyte concentrationsHowever as the sample volumesshrink to the microliter and sub-mi-croliter scale macroscopic fluores-cence methods start to lose their sen-sitivity FCS and other FFS tech-niques are microscopic fluorescencemethods The fluorescence is col-lected from a tiny fraction of the to-tal volume so reducing the overallsample volume has no detrimentaleffect on the assay We will see ex-amples where FCS related tech-niques are being used in primaryscreening applications in the sectionon fluorescence intensity distributionanalysis

At present the main disadvantageof microscopic analysis is that theassays cannot be performed in par-allel The samples must be analyzedone well at a time by scanning themicrotiter plate relative to the opticalmicroscope If it takes several sec-onds to analyze each sample thenthe total analysis time for a 2080-well plate will be on the order ofhours Although this is an acceptablelength of time in many cases thedrive for faster analysis times is everpresent New techniques for parallelimaging of the fluorescence frommultiple sample volumes are thusbeing developed to address this lim-itation3637

TWO-COLORFLUORESCENCE CROSS-CORRELATIONSPECTROSCOPY

No one chemical analysis tech-nique is able to fulfill the require-ments of every possible assay thatmight need to be performed andFCS is no exception DiffusionalFCS as we have seen only worksfor assays that involve a largechange in molecular size The boundcomplex needs to be on the order of8 times more massive than the freeligand Otherwise the different spe-cies are difficult to distinguish basedon their diffusion times alone38

Hence there has been a strong mo-

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

APPLIED SPECTROSCOPY 131A

FIG 4 A section of a 2080 sample well lsquolsquoNanoCarrierrsquorsquo plate for ultra-high throughput screening of microliter samples by FFS Thesample wells are formed from polypropylene The bottom of the carrier is 170 mm thick coverglass to allow epi-illumination of thesamples from below The carrier has dimensions of 86 3 128 3 5 mm with well volumes of 05ndash15 mL each A paper clip isshown for size comparison (Copyright Evotec Technologies)

tivation to develop alternative FFSstrategies that are sensitive to otherproperties of the system besides dif-fusion One such strategy is an FCSbased technique known as two-colorfluorescence cross-correlation spec-troscopy (2cFCCS)39ndash46 In 2cFCCSthe detection volume is formed byspatially overlapping two excitationlaser beams each operating at a dif-ferent wavelength Two different dyemolecules that absorb light in differ-ent spectral regions can both be ex-cited within the same detection vol-ume Fluorescence generated in thedetection volume is split into twodifferent detection channels eachsensitive to the emission spectrum ofone of the dyes The signals from thetwo detectors are then subjected tocross-correlation analysis Instead of

comparing signals from the same de-tector at two different times as inautocorrelation analysis the compar-ison is made between the signalsfrom detector 1 at time t and detector2 at time t 1 t The cross-correlationfunction is then obtained by inte-grating over all values of t Mathe-matically this is expressed as

T I (t)I (t 1 t)1 2G(t) 5 lim dt (9)E ^I ampmiddot^I ampTrarr` 1 20

where I1 and I2 are the fluorescencesignals from detectors 1 and 2 re-spectively The key aspect of 2cFCCSis that contributions to the cross-cor-relation function only occur whenboth fluorophors are simultaneouslypresent in the detection volume Thismeans that binding assays can be

constructed in which each bindingpartner is labeled with a differentfluorophor The binding reaction cre-ates a doubly labeled complex thatcan be detected via 2cFCCS where-as the singly labeled unbound spe-cies make no contribution By anal-ogy assays involving the decompo-sition of a doubly labeled moleculeto form two singly labeled productscan also be studied in this way Theassays do not depend on changes inmolecular size but only on the co-incident detection of both fluoro-phors Another advantage over dif-fusional FCS is that the amplitude ofthe correlation function occurring att 5 0 is directly proportional to theconcentration of the doubly labeledspecies By comparing the amplitudeof the cross-correlation function

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

132A Volume 58 Number 5 2004

focal point

GC(0) with the autocorrelation func-tion amplitudes from each detectionvolume G1(0) and G2(0) determinedfrom the same data set one can di-rectly measure the concentration ofthe doubly labeled complex using

G (0)CC 5 (10)V G (0)G (0)eff 1 2

where C is the average concentrationof the complex and Veff is the con-focal detection volume

The 2cFCCS technique is the ba-sis for a number of assays that aredirectly relevant to drug discoveryresearch at both the primary andsecondary screening level40ndash4245 Forexample Kettling et al devised anenzyme inhibition assay based onenzymatic cleavage of double-stranded DNA40 The DNA was la-beled on either end with rhodaminegreen and Cy5 fluorophors with therecognition sequence for the enzymenear the center of the strand Enzy-matic cleavage of the DNA caused adecay of the cross-correlation func-tion amplitude due to the diminishedconcentration of doubly labeled spe-cies This decay can be monitored asa function of time as well as sub-strate concentration Analysis of thecross-correlation functions was usedto measure the kinetic rate parame-ters for the enzymatic reaction andto characterize the effect of variousinhibitors As is the case with allFFS techniques these assays arecompatible with a microscopic for-mat appropriate for ultra-highthroughput screening41 In view ofthis fact Kolterman et al coined thephrase lsquolsquorapid assay processing byintegration of dual-color FCSrsquorsquo orRAPID FCS to characterize thetechnique4143

One of the difficulties of 2cFCCSis that the spatial overlap betweenthe two excitation laser beams isnever quite perfect This can causeerrors in quantifying the analyte con-centrations Offsets in the positionsof the laser beams are caused bychromatic aberration of the objectivelens as well as differences in the dif-fraction-limited size of the focal vol-umes Schwille and co-workers re-cently overcame this problem by us-

ing two-photon excitation with a sin-gle excitation laser to inducefluorescence from two different fluo-rophors Two-photon excitation oc-curs when a molecule undergoes ex-citation to its fluorescent state by si-multaneously absorbing two photonsof the same photon energy Eachphoton imparts half the energy dif-ference between the ground and ex-cited states of the fluorophor A mol-ecule that normally absorbs visibleor UV light when excited with a sin-gle photon would thus absorb in thenear-infrared via a two-photon pro-cess Electronic absorbance spectraof dyes tend to be broad and contin-uous in the UV so the same two-photon excitation wavelength cantypically be used to excite multiplefluorescent dyes The fluorescentproteins green fluorescent protein(GFP) and DsRed are examples ofchromophores that both undergotwo-photon excitation at the sameexcitation wavelength but possessspectrally distinct emission spectraHence these species are suitable la-bels for 2cFCCS analysis based ontwo-photon excitation within a sin-gle excitation volume Schwille andco-workers designed a protease as-say based on this concept in whichthe substrate consisted of a smallpolypeptide labeled at either endwith GFP and DsRed (Fig 5) Prob-lems associated with incompleteoverlap of two different detectionvolumes were eliminated The un-derlying principles of the assay weresimilar to the DNA cleavage assayof Kettling et al except that the GFPand DsRed chromophores were po-sitioned close enough together on thesubstrate to interact with each othervia fluorescence resonance energytransfer (FRET) FRET occurs whenthe excitation energy of a donorchromophore is transferred to a near-by acceptor molecule causing theacceptor to fluoresce Although thepresence of FRET complicated theanalysis it resulted in an overall en-hancement in the selectivity of theassay

FLUORESCENCE INTENSITYDISTRIBUTION ANALYSIS

Fluorescence intensity distributionanalysis (FIDA)47 also referred to as

the photon counting histogram(PCH)48 is the latest development inFFS analysis and perhaps the onethat is currently experiencing themost widespread acceptance in drugdiscovery research especially whenit comes to primary screening FIDAwas developed independently byGall and co-workers and Gratton andco-workers in 19994748 It is essen-tially a confocal microscopy basedvariation of a technique originallyproposed by Qian and Elson in 1990for analyzing the moments of thefluorescence intensity distribution inmacroscopic sample volumes4950

FIDA derives its chemical selectivityfrom differences in the molecularbrightness Qi (Eq 6) of the analytemolecules Fluorescence emittedfrom the confocal detection volumeis monitored by accumulating the de-tected photons into successive timebins of equal sampling time per binIf the duration of each bin is muchshorter than the diffusion time of themolecules through the detection vol-ume then each bin represents asnapshot of the fluorescence emittedfrom the molecules occupying thedetection volume at that particularmoment in time The fluorescencedata is histogrammed according tothe number of photons detected persampling time The shape of the his-togram is a complex function of thespatial distribution of the excitationdetection volume the analyte con-centrations and the molecularbrightnesses of the analytes It isusually necessary to calibrate theserelationships by analyzing knownstandards Once this is done theconcentrations of different analytesin an unknown sample can be deter-mined based on differences in themolecular brightness of each analyte(Fig 6)

Several variations of FIDA havebeen developed that enhance itschemical selectivity even further Forexample multiple distributions canbe obtained by analyzing the photo-count data using varying samplingtimes Molecular diffusion causesthe shape of the distribution to de-pend on the sampling time This ef-fect is ignored in conventional FIDA

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

APPLIED SPECTROSCOPY 133A

FIG 5 A protease assay combining FRET and two-photon 2cFCCS analysis (a) Sche-matic representation depicting the protease assay The substrate is a peptide labeledon either end with the fluorescent proteins rsGFP and DsRed The fluorescent proteinsundergo two-photon excitation within a single excitation volume The cleavage of thelinker region by the protease terminates both FRET and cross-correlation (b) Cross-cor-relation functions measured during the proteolytic cleavage reaction During the courseof the reaction the amplitude of the cross-correlation function gradually decreasedwhereas the corresponding diffusion times remain constant assuring the identity of thesubstrate (Adapted with permission from Ref 45 Copyright 2002 by the NationalAcademy Science of the United States of America)

by making the sampling time sosmall that the molecular motion isessentially frozen in time duringeach sampling interval By charac-

terizing the sampling time depen-dence over a large time scale (micro-seconds to milliseconds) one ex-tracts the diffusion rates of the an-

alytes in addition to their molecularbrightness values This technique isreferred to as fluorescence intensitymultiple distribution analysis (FIM-DA)51 Another alternative is two-di-mensional FIDA (2d-FIDA)52 In thismethod the fluorescence is moni-tored on two detectors each sensi-tive to different emission wave-lengths (Fig 7) or to orthogonalemission polarizations A two-di-mensional histogram is constructedaccording to the number of detectedphotons per bin for each detectionchannel The shape of the histogramdepends not only on the analyte con-centrations and molecular brightnessvalues but also on the emissionwavelengths of the fluorophors ortheir rotational anisotropies depend-ing on whether the two detectionchannels are differentiated accordingto wavelength or polarization Final-ly fluorescence intensity and lifetimedistribution analysis (FILDA) com-bines the molecular brightness infor-mation with the fluorescence life-times of the analytes53 The fluores-cence is excited using a pulsed lasersource and each detected photon isrecorded along with the elapsed timebetween the excitation pulse and thetime of detection The data is histo-grammed according to the number ofphotons per bin and the sum ofelapsed times for each bin The re-sulting histogram reveals the con-centrations molecular brightnessvalues and fluorescence lifetimes ofeach analyte FILDA is conceptuallysimilar to a related technique devel-oped by Seidel and co-workers re-ferred to as burst integrated fluores-cence lifetime (BIFL) analysis54ndash56

In short FIDA based methods havebeen devised for carrying out bind-ing assays that can exploit differenc-es in a variety of fluorescence char-acteristics making for an extremelypowerful set of capabilities LikeFCS FIDA based methods are ame-nable to microscopic assays and aretherefore useful for ultra-highthroughput screening on the micro-liter scale

Fluorescence intensity distributionanalysis is particularly suitable forcharacterizing the binding of ligands

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

134A Volume 58 Number 5 2004

focal point

FIG 6 Fluorescence-intensity distribution analysis of tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G) The left figure showsthe measured distributions of photon counts for three solutions 05 nM Rh6G 15 nM TMR and a mixture of 08 nM TMR and 01nM R6G R6G has a larger molecular brightness than TMR at the excitation wavelength employed This results in a broader fluores-cence intensity distribution for the R6G sample The width of the distribution for the mixture is intermediate between that of the pureTMR and R6G samples and depends on the relative concentrations of the two components in the mixture The right figure showsresults from the analysis of the distribution functions shown on the left Dashed lines correspond to the analysis of the pure solu-tions and the solid line results from analysis of the mixture (Adapted with permission from Ref 47 copyright 1999 NationalAcademy of Sciences of the United States of America)

to membrane-bound receptor pro-teins These types of assays are im-portant in drug discovery researchbecause many of the receptors beingtargeted by drugs are cell membraneproteins Binding affinity studies areused to assess the ability of a drugcandidate to bind the receptor or toinhibit the receptorrsquos ability to bindits ligand These types of assays areproblematic for conventional fluores-cence-based methods such as FPdue to the extremely slow rotationaldiffusion of the bound ligands Theyare also difficult for diffusional FCSbecause the time needed to measuresuch slow linear diffusion rates isgenerally too lengthy for high-throughput screening purposesHowever these assays are perfectlysuited to FIDA because multiple flu-orescently labeled ligands can bindto different receptor sites on themembrane causing an enormous in-crease in the effective molecularbrightness If multiple fluorophorsare attached to the same membranethen their motion is coupled to thatof the membrane A membrane withmultiple bound fluorophors is ana-lyzed as if it were an independent

chromophore with a molecularbrightness equivalent to the totalbrightness of all the attached fluo-rophors This creates an enormousdifference between the brightness ofthe bound complex and that of thefree ligands making it easy to dis-criminate the bound complex basedon FIDA analysis Scheel et al re-cently demonstrated these conceptsby using FIDA to study the bindingof fluorescently labeled epidermalgrowth factor a polypeptide hor-mone to the epidermal growth factorreceptor which is a receptor proteinbound to the membrane of humanskin cells57 If the skin cells are can-cerous then the goal of such studieswould be to identify a drug that in-hibits this binding interaction on thecancerous cell membrane Scheel etal used FIDA to measure the bind-ing affinity of the ligand the expres-sion level of the receptor proteinand the ability of various moleculesto inhibit the binding activity of thereceptors They also showed thatthese assays could be performed onmicroliter sample volumes and on atime scale of seconds per assay

Fluorescence intensity distribution

analysis techniques are also usefuleven when there is no change in themolecular brightness or when thechange is relatively small For ex-ample the version of 2d-FIDA thatdifferentiates the two detection chan-nels according to emission polariza-tion can essentially do everythingconventional FP can do and moreLike FP it can distinguish bound andunbound ligands based on their dif-ferent fluorescence anisotropies Itcan also monitor differences in mo-lecular brightness and it can do allof this in the microscale assay for-mat Wright et al reported a directside-by-side comparison of FP and2d-FIDA for characterizing the fluo-rescence anisotropies of fluorescent-ly labeled synthetic peptides bindingto a protein involved in the mitogen-activated protein kinase pathwayone of the key processes in cellularapoptosis58 FP and 2d-FIDA gaveequivalent results for these assaysHowever the FP measurements werecarried out in more conventional384-well sample plates whereas 2d-FIDA could be performed on 1 mLsamples contained in a 1536-wellplate This resulted in a nearly 10-

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

APPLIED SPECTROSCOPY 135A

FIG 7 Spectrally resolved 2dFIDA for monitoring the binding of fluorescently labeledsomastatin molecules (green triangles) to membrane vesicles containing the somastatinreceptor The vesicles depicted as a red oval were stained with a red fluorescent lipo-philic tracer Fluorescence was monitored on two detectors sensitive to the green fluo-rescence from the ligand (detector 2) and the red fluorescence from the vesicles (detec-tor 1) respectively The two-dimensional histogram displays the number of detectedphotons per bin from each detector When the binding affinity is high the intensitydistribution is skewed toward the right side of the diagonal white line drawn throughthe histogram indicative of a high relative molecular brightness of the bound ligandsHence the shape of the histogram can serve as a measure of the fraction of boundligands (Adapted with permission from Ref 52 copyright 2000 Biophysical Society)

fold reduction in the sample volumeof each sample in going from 386 to1536 wells Performing these typesof assays on such minute quantitiesof sample will greatly enhance theability to screen larger and largercompound libraries while holding

the costs of the reagents needed toperform the assays to a minimum

COMMERCIALINSTRUMENTATION

An important criterion for the evo-lution of a technique into a mature

chemical analysis tool used in prob-lem-driven research is that it be-comes accessible to non-specialistsOne way in which this can happen iswhen effective lsquolsquouser-friendlyrsquorsquocommercial instrumentation for per-forming the desired measurementsbecomes available Several commer-cial FFS instruments are availablethat meet this criteria which is an-other good indication that thesemethods are becoming widely ac-cepted in a broad range of fieldsThese instruments can be catego-rized into those that are used pri-marily for detailed biophysical in-vestigations of specific compounds(ie for secondary screening) andthose that are dedicated to primaryscreening of large compound librar-ies The first commercial FFS instru-ment was the ConfoCorr FCS spec-trometer This instrument was devel-oped in 1993 through a collaborativeeffort between Carl Zeiss Jena (JenaGermany) and EVOTEC Biosystems(Now EVOTEC Technologies Ham-burg Germany) The originalConfoCorr performed detailed FCSanalysis of individual compounds orsmall compound libraries but it onlysupported a single excitation laserbeam and a single detection channelfor doing conventional diffusionalautocorrelation measurements A lat-er design the ConfoCorr 2 first ap-peared in 1997 and is still beingmanufactured and sold by Carl ZeissJena as a research-grade FCS instru-ment for detailed biophysical inves-tigation59 The ConfoCorr 2 allowsboth single beam autocorrelationanalysis and two-channel excitationand detection for FCCS analysis Itincludes a laser module that allowsswitching between one or two dif-ferent excitation wavelengths and alaser scanner for FCS based imagingof cells and tissues with submicro-meter spatial resolution

Another example of a research-grade commercial instrument usedprimarily for secondary screeningand other biophysical measurementsis the Insight from EVOTEC Thisinstrument utilizes the lsquolsquoFCS1plusrsquorsquoconcept6 FCS1plus supports a suiteof molecular analysis capabilities

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

136A Volume 58 Number 5 2004

focal point

FIG 8 The Clarina from Evotec Technologies is an example of a commercial FFS in-strument currently being used for high-throughput primary screening applications Thelower left photo shows a microtiter plate scanner above an inverted optical micro-scope objective The upper left photo shows a 96-well microtiter plate mounted in thescanner The upper right photo shows a close-up of the microscope objective used tofocus the excitation beam and collect fluorescence from each sample (courtesy of Evo-tec Technologies)

including all of the main FFS meth-ods (FCS FCCS FIDA FIMDA2d-FIDA and FILDA) It also in-cludes some of the more convention-al fluorescence techniques such asresonance energy transfer quench-ing anisotropy and lifetime analy-sis all combined with sub-micro-meter fluorescence imaging capabil-ities Other instruments that incor-porate the FCS1plus conceptinclude the Clarina II (Fig 8) theDA20 and the EVOScreen systemsfrom EVOTEC These instrumentsare used in automated primaryscreening of multiple compoundsAll of these instruments areequipped with fully automatedFCS1plus readers and sample scan-ning and data acquisition electronicsThe EVOScreen instrument also uti-lizes an advanced liquid handlingsystem for creating large-scale arrays

of samples with microliter samplevolumes in an automated fashionThese instruments are capable ofperforming hundreds to thousands ofassays with total analysis times inthe range of tens of minutes to hoursA number of major pharmaceuticalcompanies have begun to adopt thistechnology in recent years

CONCLUSION

Fluorescence fluctuation spectros-copy is a technique that is coming ofage as a mature chemical analysistool This is occurring in a variety ofways We have emphasized the tre-mendous advantages these methodsimpart in the field of drug discoveryLigandndashreceptor binding assays canbe constructed that monitor a widerange of molecular characteristicsincluding changes in diffusion prop-

erties fluorescence anisotropy emis-sion wavelength lifetime and fluo-rescence efficiency These assays canbe performed in situ on samples ofonly a microliter in volume or lessand with sufficient analysis speed toallow hundreds to thousands of as-says to be carried out in a reasonabletime frame (ie minutes to hours)FFS methods are already starting toimpact the drug discovery field andthis trend will likely continue longinto the future as the number of po-tential drug targets increases at a rap-id pace FFS is influencing manyother areas of research in addition todrug discovery A few examples in-clude the characterization of photo-physical and photochemical process-es60 biomolecular conformationaldynamics6162 adsorptiondesorptionand molecular diffusion at solidndashliq-uid interfaces and biological mem-branes63ndash66 molecular flow profilingin microfluidics devices67ndash69 multi-component electrophoretic analysis7071 and intracellular molecular dy-namics and imaging72 In many ofthese examples FFS is providingcrucial new insight into the nature ofthe system that would be difficult orimpossible to attain in any otherway Hence we can expect thesetechniques to continue maturing asthey follow the path toward becom-ing indispensable tools in biomolec-ular research Jean Perrin would beamazed to see how far we have comefrom the days when the only way toanalyze molecular motion was tolook through a microscope andpainstakingly record the displace-ments of microscopic particles oneparticle at a time

ACKNOWLEDGMENTS

We thank Martin Daffertshofer of EVOTECTechnologies Klaus Weisshart of Karl ZeissJena GmbH and Edmund Matoyashi of Ab-bott Laboratories for their assistance with thisarticle We thank Petra Schwille and TobiasKohl for providing Figure 5 FFS research be-ing carried out in the authorsrsquo laboratory issupported by the National Institutes of Health-National Center for Research Resources(Grant Number RR17025)

1 D Magde W W Webb and E L ElsonPhys Rev Lett 29 705 (1972)

2 R Rigler and E S Elson Eds Fluores-cence Correlation Spectroscopy Theory

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