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German Edition: DOI: 10.1002/ange.201501949Super-Resolution MicroscopyInternational Edition: DOI: 10.1002/anie.201501949

Single-Molecule Spectroscopy, Imaging, andPhotocontrol: Foundations for Super-ResolutionMicroscopy (Nobel Lecture)**W. E. (William E.) Moerner*

fluorescence microscopy · photocontrol ·single-molecule spectroscopy · super-resolution

1. The Early Days

1.1. Introduction and Early Inspirations

I want to thank the Nobel Committee for Chemistry, theRoyal Swedish Academy of Sciences, and the Nobel Founda-tion for selecting me for this prize recognizing the develop-ment of super-resolved fluorescence microscopy. I am trulyhonored to share the prize with my two esteemed colleagues,Stefan Hell and Eric Betzig. My primary contributions centeron the first optical detection and spectroscopy of singlemolecules in the condensed phase,[1] and on the observationsof imaging, blinking and photocontrol not only for single

molecules at low temperatures insolids, but also for useful variantsof the green fluorescent proteinat room temperature.[2] This lec-ture describes the context of theevents leading up to these advan-ces as well as a portion of thesubsequent developments botharound the world and in mylaboratory.

In the mid-1980s, I derivedmuch early inspiration fromamazing advances that wereoccurring around the worldwhere single nanoscale quantumsystems were detected and

explored for both scientific and technological reasons. Someof these were 1) the spectroscopy of single electrons or ionsconfined in vacuum electromagnetic traps,[3–5] 2) scanningtunneling microscopy (STM)[6] and atomic force microscopy(AFM),[7] and 3) the study of ion currents in single mem-brane-embedded ion channels.[8] But why not optical detec-

The initial steps toward optical detection and spectroscopy of singlemolecules in condensed matter arose out of the study of inhomoge-neously broadened optical absorption profiles of molecular impuritiesin solids at low temperatures. Spectral signatures relating to the fluc-tuations of the number of molecules in resonance led to the attainmentof the single-molecule limit in 1989 using frequency-modulation laserspectroscopy. In the early 90s, many fascinating physical effects wereobserved for individual molecules, and the imaging of single moleculesas well as observations of spectral diffusion, optical switching and theability to select different single molecules in the same focal volumesimply by tuning the pumping laser frequency provided importantforerunners of the later super-resolution microscopy with singlemolecules. In the room temperature regime, imaging of single copies ofthe green fluorescent protein also uncovered surprises, especially theblinking and photoinduced recovery of emitters, which stimulatedfurther development of photoswitchable fluorescent protein labels.Because each single fluorophore acts a light source roughly 1 nm insize, microscopic observation and localization of individual fluo-rophores is a key ingredient to imaging beyond the optical diffractionlimit. Combining this with active control of the number of emittingmolecules in the pumped volume led to the super-resolution imaging ofEric Betzig and others, a new frontier for optical microscopy beyondthe diffraction limit. The background leading up to these observationsis described and current developments are summarized.

[*] Prof. Dr. W. E. MoernerDepartments of Chemistry and (by Courtesy) of Applied Physics,Stanford UniversityStanford, California 94305 (USA)

[**] CopyrightÓ The Nobel Foundation 2014. We thank the NobelFoundation, Stockholm, for permission to print this lecture.

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tion and spectroscopy of a single small molecule deep insidea more complex condensed phase environment than ina vacuum, which would enable single-molecule spectroscopy(SMS)?

There was a problem. Years before, the great theoreticalphysicist and co-founder of quantum mechanics, ErwinSchrçdinger stated:[9]

“…we never experiment with just one electron or atom or(small) molecule. In thought-experiments we sometimesassume that we do; this invariably entails ridiculous conse-quences…In the first place it is fair to state that we are notexperimenting with single particles, any more than we can raiseIchthyosauria in the zoo.”

Schrçdinger was not the only one who felt this way, evenin the 1980s. Many scientists believed that, even though singlephotoelectrons might be detected from photoionization ofa single molecule in a vacuum, for example, opticallydetecting a single molecule in a condensed phase samplewas impossible. Thus the key aspect of the early part of thisstory is to explain how I got to the point to believe that itwould be possible.

1.2. Low Temperature Spectroscopy of Molecules in Solids:Inhomogeneous Broadening

In order to explain the initial SMS experiments in the late1980s, it is necessary to briefly review some concepts fromhigh resolution optical spectroscopy of molecular impuritiesin solids, a field of intense study in the decades surrounding1970 driven by names such as E. V. ShpolÏskii, R. Personov,K. K. Rebane, and others. (Exhaustive references cannot beincluded here, but for a comprehensive text covering manyaspects, see Ref. [10a].) Beginning at room temperature, letus consider the optical absorption spectrum of terrylenemolecules dispersed at low concentration (say 10¢6 molmol¢1)in a solid transparent host of p-terphenyl (see Figure 1). Thefigure shows the optical absorption expressed in opticaldensity of a sample versus the wavelength l of light used forprobing, the kind of spectrum one can obtain from a commer-

cial UV/Vis spectrometer. A color scale shows the corre-spondence to the colors of visible light. Starting with longwavelengths on the right, there is no absorption, and as l getsshorter (and energy gets higher according to E = hf with hPlanckÏs constant, f frequency), eventually the moleculeabsorbs light. The arrow shows the first electronic transitionrepresenting the promotion of an electron from the groundstate (highest occupied molecular orbital) to the first excitedstate (lowest unoccupied molecular orbital). The shorterwavelength absorptions shown involve the creation of addi-tional vibrations in the molecule. In addition, since f and l areinversely related (f = c/l with c the speed of light), ifl increases to the right, then frequency increases to the left.The frequencies at the edges of the plot are shown, in therange of hundreds of THz (1012 cycles per second).

Terrylene (and other similar aromatic hydrocarbons) isa relatively planar, rigid molecule which is held flat by theconjugated p orbitals of the molecule and the bonds that aredenoted by the aromatic rings of the molecule. Because ofthis, the first electronic transition does not involve largedistortions of the molecule, that is, this transition involvesprimarily the electronic degrees of freedom of the molecule,also termed minimal Franck–Condon distortion. Now letÏscool the sample to very low temperatures of a few Kelvinabove absolute zero (liquid helium temperatures), andexpand the horizontal scale by roughly 25 times. Spectro-scopists often switch between wavelength and frequencydisplays, and Figure 2 now shows frequency increasing to the

right, in units of wavenumber or inverse cm; 1 cm¢1 corre-sponds to roughly 30 GHz. Only a small piece of orangewavelengths is left. At such low temperatures, the vibrationsof the molecule cannot be thermally excited, so the appear-ance of the first electronic transition is now extremely narrow.Moreover, the vibrations of the solid (phonons) are essen-tially nonexistent too, so they cannot contribute to broad-ening of the optical absorption, and the line becomes a “zero-phonon” line. In fact, in the p-terphenyl host crystal, there arefour inequivalent locations for the terrylene molecules in the

Figure 1. Spectrum (absorption versus wavelength, or color) of terry-lene molecules in a solid host of p-terphenyl at room temperature.

Figure 2. Spectrum (absorption versus frequency, wavenumber, orcolor) of terrylene molecules in a solid host, p-terphenyl, at lowtemperature (�2 K), from Ref. [10b].

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W. E. Moerner

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crystal, thus there are four electronic “origins”: X1–X4,because the structure of the host in these four different sitesare quite different. The absorption lines have become narrowenough so that the different perturbations coming from thedifferent local environments (think local pressure) are nowobservable.

In fact the spectrum in Figure 2 does not tell the wholestory, because the width of the absorption line for any one ofthe four sites is far narrower than shown. To fully resolve theabsorption line in a way not limited by the apparatus,spectroscopists began to use narrowband dispersing devicessuch as double monochromators or ultimately single fre-quency continuous wave (cw) tunable lasers as light sources,but a further surprise was waiting. Figure 3a shows the

situation for pentacene dopant molecules in p-terphenylcrystals at 1.8 K as reported by Orlowski and Zewail in1979.[11] One might expect that now the linewidth should beonly about 10 MHz or so, the expected width for themolecular absorptions if it were limited only by the lifetimeof the excited state. However, the absorption profile is muchwider, about 0.7 cm¢1 or about 21 GHz! This excess width wasrecognized as inhomogeneous broadening as schematized inFigure 3b. The various molecules in the solid have intrinsi-cally narrow widths (called the homogeneous width), but theoverall absorption line profile represents the range of differ-ent center frequencies for the molecules arising from differentlocal environments (illustrated in Figure 3c) which shift theabsorption energies over a range. These perturbations arisefrom effects like local stresses and strains arising from crystalimperfections, or from other defects, or possibly from localelectric fields, and so on, and a number of theoretical modelswere proposed for the mechanisms of inhomogeneous broad-ening.[12]

1.3. The Environment at IBM Research: Spectral Hole-Burningfor Optical Storage

One goal of high-resolution spectroscopy was to measurethe true homogeneous width of the zero phonon, purelyelectronic transition of molecules in solids without theinterference from inhomogeneous broadening. It is for this

reason that much research in the 1970s and 1980s was devotedto methods like fluorescence line narrowing (FLN)[13, 14] andtransient spectroscopies such as free induction decay, opticalnutation, and photon echoes.[15–17] While these were allpowerful methods with advantages and disadvantages, therewas another method to assess the homogeneous width undercertain circumstances, persistent spectral hole-burning, illus-

trated in Figure 4. This optical effect was discovered in the1970s by two Russian groups: by Gorokhovskii et al. for H2-phthalocyanine in a ShpolÏskii matrix,[18] and by Kharlamovet al. for perylene and 9-aminoacridine in glassy ethanol.[19]

Persistent spectral hole-burning turned out to be a fairlycommon effect in the optical transitions of impurities in solidsat low temperatures. Given an inhomogeneously broadenedline (Figure 4 upper), irradiation with a narrowband laseronly excites the subset of molecules resonant with the laserwithin a homogeneous width GH. Spectral hole-burning occurswhen light-driven physical or chemical changes are producedonly in those molecules resonant with the light, driving thesemolecules to some other part of frequency or wavelengthspace. This leaves behind a dip or “spectral hole” in theoverall absorption profile of width roughly 2GH. Importantly,it was realized by scientists at IBM Research that hole-burning may be used for optical recording of information inthe optical frequency domain, hence the term “frequencydomain optical storage”.[20] For more details on spectral hole-burning see Ref. [21].

In 1981, I joined one of the great corporate research labsat the time, IBM Research, to work on materials andmechanisms for spectral hole-burning storage. This wasa time where a novel idea with potential application couldbe studied in great detail in a corporate research lab, from the

Figure 3. Inhomogeneous broadening in solids at low temperatures.a) Pentacene in p-terphenyl absorption spectra taken from Ref. [11](0.09 cm¢1 resolution). b) Schematic of the inhomogeneous broad-ening effect with width GI. c) Schematic of different local environmentsgiving rise to inhomogeneous broadening.

Figure 4. Illustration of persistent spectral hole-burning in inhomoge-neous broadened lines of dopants in solids at low temperatures.a) Before burning, the inhomogeneously broadened line hasa “smooth” absorption profile. b) When a narrowband laser irradiatesselected frequencies in certain materials, spectral dips or “holes” canbe generated.


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fundamental scientific issues to thedevelopment of the required materials,to the potential engineering design ofthe system. Persistent spectral hole-burning was of interest because itwould enable many bits of informationto be stored in the same spot in theoptical frequency domain simply bychoosing to either write a hole or notwrite a hole in the inhomogeneouslybroadened line profile. Since fora number of systems the ratio GI/GH

was on the order of 1000 or more atlow temperatures, a huge increase inoptical storage capacity was envi-sioned. Mechanisms for the processcould be photochemical[22] where thelight induces a photochemical change,or photophysical (nonphotochemical), where only the two-level systems of the nearby host need be changed,[23, 24] andmuch effort centered on the generation of new materialssystems. Unfortunately, in the end the need for low temper-atures and the amazing compound growth rate of magneticstorage performance squeezed this idea out of practicalapplication, although microwave signal processing applica-tions[25] are still being explored using hole-burning effects.

1.4. Statistical Fine Structure in Inhomogeneously BroadenedLines

Luckily, it was also important at IBM to examine thefundamental limits to new technologies for optical storage,and this was particularly interesting to me. In 1985, I workedwith Marc Levenson on the shortcomings of one-photon(linear) hole-burning mechanisms.[26] As spectral holes arewritten at higher and higher speed, the actual depth of thehole will get smaller up until it has a fractional depth equal tothe one-cycle quantum efficiency for spectral hole formation.In addition to shot noise due to Poisson number fluctuationsof the probing light, we realized that a particularly interestingadditional limitation on the signal-to-noise ratio of a spectralhole might result from the finite number of molecules thatcontribute to the absorption profile near the hole. Thequestion arose: Is there a static spectral roughness on theinhomogeneous line that results from statistical numberfluctuations or the discreteness of individual molecules? Thiswould define one ultimate limit on the smallest spectral holethat could be detected. The basic idea is illustrated fromfamiliar probability considerations in Figure 5. Supposing that50 balls are thrown randomly at 10 bins, it is quite unlikelythat exactly 5 balls will land in each bin (Figure 5 a). Rather,a much more likely outcome of a single experiment is shownin Figure 5b: the numbers landing in each bin will have anaverage value of 5 over all bins, but the actual numbers willscatter above and below this value. This is the familiarnumber fluctuation effect, equivalent to the scaling of thestandard error of the mean, where the rms size of thefluctuations about the mean will scale as

pN, where N is the

average number, orp

5 in this case. The central limit theoremapplies here since the molecules are assumed independent.

Now to see how this idea relates to high resolutionspectroscopy of inhomogeneously broadened lines at lowtemperatures, we simply think of the horizontal axis as opticalfrequency (or wavelength), and imagine that GI is extremelylarge so that the inhomogeneous line appears locally flat onthe scale of the page. Each box is a bin of width GH, thehomogeneous width of an optical absorption line, andmolecules pick frequencies when the sample is formed ina random way. Then the resulting spectrum should havea spectral roughness or fine structure scaling as

pN, which

arises from the discreteness of the individual molecules! Thiscan also be seen in the simulation of Figure 5c, wherea (perfectly smooth) Gaussian shape was assumed for theprobability of molecules to assume specific resonance fre-quencies. At very small numbers of total molecules, thevariations in absorption are obvious, and at larger and largerconcentrations, the effect appears to smooth out, as was likelythe case in the early spectra of pentacene in p-terphenyl inFigure 3a.[11] (In addition, the spectral resolution was too lowto see this effect in the early experiments.) It is critical to notethat this effect is not “noise” in the usual sense of time-dependent interfering fluctuations, but rather a static varia-tion in absorption versus wavelength or optical frequency. Wenamed this effect “statistical fine structure” (SFS), and it isimportant to realize that the relative size of SFS gets smaller athigh concentration as 1

� ffiffiffiffiffiffiffiNH

p¨ ¦, while the absolute root-

mean-square (rms) size of the fine structure grows asffiffiffiffiffiffiffiNH

p.

Surprisingly, prior to the late 1980s, the observation of SFShad not been reported, so this was a first goal.

In 1987, my postdoc Tom Carter and I observed SFS forthe first time,[27, 28] using a powerful zero-background opticalabsorption technique, laser frequency-modulation (FM)spectroscopy,[29, 30] explained below. The choice of samplewas critical: We actually tried for many months to see theeffect for perylene dopant molecules in a thin film ofpoly(vinyl chloride), but each time we scanned the spectrumand saw a hint of the structure, it changed for the next scan!This was due to photophysical hole-burning for this systemcaused by the probing laser. At one point, in frustration due to

Figure 5. Illustration of number fluctuations for probability and for spectroscopy. a) An unlikelyway to randomly throw 50 balls at 10 bins. b) A more likely case. c) Simulation of aninhomogeneously broadened line in a sample for the case of uniform Gaussian probability ofselecting absorption frequencies.


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the need for a system with no hole-burning, I consultedMichael Fayer at Stanford, who suggested pentacene dopantmolecules in a p-terphenyl crystal (Figure 6, left). Over theweekend, I simply melted some p-terphenyl laced with a tinyspeck of pentacene on a hot plate between glass slides, put itin the cryostat, and we immediately saw the SFS signal!Figure 6, right, shows a small 5 GHz slice of the O1 siteinhomogeneous line centered roughly at 506 THz. SFS is theamazing spectral structure which repeats beautifully when thescan is repeated (upper panel). SFS is clearly unusual, in thatits size depends not upon the total number of resonantmolecules, but rather upon the square root of the number, andit arises directly from the discreteness of the individualmolecules. (It turns out that hole-burning was not completelyabsent in this system, but only far less probable—withextended laser irradiation, spectral holes could be burneddirectly in the SFS.)

2. Single-Molecule Detection, Spectroscopy, andImaging at Low Temperatures

2.1. FMS and a Scaling Argument Led the Way to the First Single-Molecule Detection and Spectroscopy in Condensed Phases

The other crucial aspect of the SFS experiment was theultrasensitive optical detection method used, laser FMspectroscopy (FMS).[29,31] FMS was invented by Gary C.Bjorklund at IBM in 1980, and he taught me this method to beable to use it for detection of spectral holes. As illustrated inFigure 7, a single-frequency tunable laser at frequency wC

passes through an electro-optic phase modulator and acquiresfrequency modulation at an rf modulation frequency wM,usually on the order of 100 MHz. In the frequency domain,

two sidebands appear as shown. Thesesidebands are out-of-phase, so that if theyare not disturbed by the sample, a fastdetector which naturally measures theenvelope of the light wave produces nosignal at wM. However, when a narrowspectral feature (narrow on the scale of wM)is present, the imbalance in the laser side-bands leads to amplitude modulation in thedetected photocurrent at wM which is easilydetected by rf lock-in techniques. In otherwords, the sample converts the FM beaminto an AM beam when a narrow feature ispresent, and the whole experiment behavesroughly like FM radio at 506 THz (albeit atlow modulation index). A key feature ofFMS is that it senses only the deviations ofthe absorption from the average value;more precisely, the signal is proportional toa(wC + wM)¢a(wC¢wM) with a the absorp-tion coefficient. This is the main reasonwhy the detection of SFS could be easilyaccomplished with FMS. There was noneed to make an heroic sample with ultra-low concentration to see SFS, because the

SFS signal measured by FMS is actually larger with higherconcentrations of molecules!

Nevertheless, with the detection of SFS in hand, it nowbecame possible for me to believe that single-moleculedetection would be possible. This key point can be understoodby a simple scaling argument: When SFS due to approximately1000 molecules is detectable (roughly the case in Figure 6),that means that the measured FMS signal (rms amplitude) isthe same size as approximately 32 molecules (i.e., (1000)1/2).But this means that in terms of improving the signal-to-noiseratio (SNR) of the FMS apparatus, it was only necessary towork 32 times harder to observe a single molecule, not 1000times harder! This realization, combined with two additionalfacts: 1) FM absorption spectroscopy was insensitive to anyRayleigh or Raman scattering background from imperfectsamples, and 2) FMS allows quantum-limited detection sensi-tivity, led me and my postdoc Lothar Kador (Figure 8) to pushFM spectroscopy to the single-molecule limit. It is also truethat the particularly low quantum efficiency for spectral hole-burning made pentacene in p-terphenyl an excellent firstchoice for single-molecule detection.

The first SMS experiments in 1989 utilized either of twopowerful double-modulation FMS absorption techniques,laser FMS with Stark secondary modulation (FM-Stark) orFMS with ultrasonic strain secondary modulation (FM-US).[1,32] Secondary modulation was required in order toremove the effects of residual amplitude modulation producedby the imperfect phase modulator.[33] Figure 8 (specifically,trace d) shows examples of the optical absorption spectrumfrom a single molecule of pentacene in p-terphenyl using theFM-Stark method, where the laser center frequency wassimply tuned into the wings of the inhomogeneously broad-ened line in order to select the single-molecule concentrationrange without growing a new sample with reduced doping.

Figure 6. Observation of statistical fine structure (SFS) (right) for pentacene in p-terphenyl(schematic structure) with Tom Carter (photo). Data from Ref. [28].


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Figure 7. Laser frequency-modulation spectroscopy for detection of weak absorption and dispersion signals. Photo: Gary C. Bjorklund. FromRef. [33].

Figure 8. First optical detection and spectroscopy of single molecules in condensed matter. a–c) Buildup of expected lineshape for FM-Starkspectroscopy. d) Multiple scans showing a single molecule at 592.423 nm. e) Averaged scans compared to lineshape. f) Far into the wings of theline, no molecule. g) Closer to the center of the inhomogeneous line; SFS. Photo: Lothar Kador. From Ref. [1].


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Although this early observation and similar data from theFM-US method served to stimulate much further work, therewas one important limitation to the general use of FMmethods for SMS. As was shown in the early papers onFMS,[29, 31] extremely weak absorption features as small as 10¢7

in relative size can be detected in a 1 s averaging time, butonly if large laser powers on the order of several mW can bedelivered to the detector to force the photon shot noise todominate detector Johnson noise. However, in SMS, the laserbeam must be focused to a small spot to maximize the opticaltransition probability, thus the power in the laser beam mustbe maintained below the value which would cause saturationbroadening of the single-molecule lineshape, which is hard toavoid for such a narrow line at low temperatures. As a result,the data of Figure 8 had to be acquired with powers below100 mW at the detector, which is one reason why the SNR wasonly on the order of 5. (The other reason was the use ofrelatively thick cleaved samples, which produced a populationof weaker out-of-focus molecules in the probed volume. Thisproblem was subsequently easily overcome.) In later experi-ments by Lothar,[34] frequency modulation of the absorptionline itself (rather than the laser) was produced by anoscillating (Stark) electric field alone, and this method hasalso been used to detect the absorption from a single moleculeat liquid helium temperatures. While successful, these trans-mission methods are limited by the quantum shot noise of thelaser beam, which is relatively large at the low laser intensityrequired to prevent saturation.

2.2. Crucial Milestone: Detection of Single-Molecule Absorptionby Fluorescence

The optical absorption experiments on pentacene in p-terphenyl indeed showed that this material has sufficientlyinefficient spectral hole-burning to make it a useful modelsystem for single-molecule studies. In 1990, Michel Orrit andJacky Bernard demonstrated that sensing the optical absorp-tion by detection of the emitted fluorescence producessuperior signal-to-noise if the emission is collected efficientlyand the scattering sources are minimized.[35] Due to itsrelative simplicity, subsequent experiments have almostexclusively used this method, which is also called “fluores-cence excitation spectroscopy.” It is an application of the gas-phase method of laser-induced fluorescence pioneered byR. N. Zare in 1968[36] to solids. In fluorescence excitation,a tunable narrowband single-frequency laser is scanned overthe absorption profile of the single molecule, and the presenceof absorption is detected by measuring the fluorescenceemitted (Figure 9) to long wavelengths, away from the laserwavelength itself. The method is often background-limited,and it requires the growth of ultrathin crystal clear sublimedflakes to reduce the scattering signals that could arise fromthe p-terphenyl crystal, but it does not suffer from the difficulttradeoff between SNR and optical broadening of FMS. Thiswas a major advance for single-molecule spectroscopy, and ifthere were a fourth recipient for the Nobel Prize, Michel Orritshould have received it.

Figure 9. Single-molecule detection and absorption spectroscopy by recording the emitted fluorescence. A,B) The inhomogeneous line, C) at verylow concentration; the dots represent the increases in emission when single molecules come into resonance with the tunable laser. Photo: MichelOrrit. From Ref. [35].


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With the ability to detect single molecules in crystals andpolymers, in the early 1990s many investigators all over theworld jumped into the field in order to take advantage of theextremely narrow optical absorption lines and the removal ofensemble averaging, two of the largest motivations for thestudy of single molecules. Investigations were sometimesdirected at specific observations of particular effects like theStark effect,[37] two-level system dynamics,[38] or polarizationeffects[39] to name a few. At other times experiments wereperformed simply to observe, because surprises would beexpected when a new regime is first explored. The great bodyof work done is too large to review here, and the reader isreferred to selected texts[21,40] and selected review articles[41–47]

for more information. My talented group of postdocs andcollaborators completed a wide array of experiments, includ-ing measurements of the lifetime-limited width, temperature-dependent dephasing, and optical saturation effects,[48,49]

photon antibunching correlations,[50] vibrational spectrosco-py,[51–53] magnetic resonance of a single molecular spin,[54] andnear-field spectroscopy.[55] Some experiments have particularrelevance for super-resolution microscopy and will be dis-cussed next.

2.3. Single-Molecule Spectroscopy and Imaging

With the single-molecule sensitivity that became availablein the early 1990s, a more detailed picture of inhomogeneousbroadening appeared. Figure 10 a shows a scan over the

inhomogeneously broadened optical absorption profile forpentacene in p-terphenyl; compare Figure 3 a. While mole-cules overlap near the center of the line at 592.321 nm(0 GHz), in the wings of the line, single, isolated Lorentzianprofiles are observed as each molecule comes into resonancewith the tunable laser. The situation is very much like tuningyour AM radio to find a station while far away from big cities:you tune and tune, mostly hearing static, until you come intoresonance with a station, then the signal rises above the static.It is obvious that with a lower-concentration sample, singlemolecules at the center of the inhomogeneous like could alsobe studied, and the line profile is only Gaussian near thecenter—there are large non-Gaussian tails quite far awayfrom the center. These beautiful spectra provided much of thebasis for the early experiments. For example, at low pumpingintensity, the lifetime-limited homogeneous linewidth of 7.8�0.2 MHz was directly observed (Figure 10b).[56] This linewidthis the minimum value allowed by the lifetime of the S1 excitedstate of 24 ns, in excellent agreement with previous photonecho measurements on large ensembles.[15,57] Such narrowsingle-molecule absorption lines are wonderful for thespectroscopist: many detailed studies of the local environ-ment can be performed, because narrow lines are much moresensitive to local perturbations than are broad spectralfeatures.

Going beyond spectral studies alone, a hybrid image ofa single molecule was obtained by Pat Ambrose in my lab byacquiring spectra as a function of the position of the laserfocal spot in the sample.[48] Spatial scanning was accomplished

Figure 10. a,b) Single-molecule spectroscopy and c,d) imaging. Photos: W. Pat Ambrose (upper), Urs P. Wild. From Refs. [49] (a),[56] (b), [48] (c),[58] (d).


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in a manner similar to confocal microscopy by scanning theincident laser beam focal spot across the sample in one spatialdimension. Figure 10 c shows such a three-dimensional“pseudo-image” of single molecules of pentacene in p-terphenyl. The z-axis of the image is the (red-shifted)emission signal, the horizontal axis is the laser frequencydetuning (300 MHz range), and the axis going into the page isone transverse spatial dimension produced by scanning thelaser focal spot (40 mm range). In the frequency domain, thespectral features are fully resolved because the laser linewidthof ca. 3 MHz is smaller than the molecular linewidth.However, considering this image along the spatial dimension,the single molecule (SM) is actually serving as a highlylocalized nanoprobe of the laser beam diameter itself (hereca. 5 mm, due to the poor quality of the focus produced by thelens in liquid helium).[48] The molecule be regarded asa nanoscale (nm-sized) probe of the focal spot, which isroughly equivalent to a measurement of the point-spread-function (PSF) of the imaging system. This is the first exampleof a spatial image of a single molecule PSF, discussed in moredetail below. Soon thereafter, the Wild laboratory in Switzer-land[58] obtained two-dimensional images of the shape ofa single-molecule spot as shown in Figure 10d in reversecontrast.

2.4. Surprises—Spectral Diffusion and Optical Control

During the early SMS studies on pentacene in p-terphenyl, an unexpected phenomenon appeared: resonancefrequency shifts of individual pentacene molecules in a crystalat 1.5 K,[48, 56] mentioned briefly earlier.[35] We called this effect“spectral diffusion” due to its close relationship to similarspectral-shifting behavior long postulated for optical transi-tions of impurities in amorphous systems.[59] Here, spectraldiffusion means changes in the center (resonance) frequencyof a single molecule due to configurational changes in thenearby host which affect the frequency of the electronictransition via guest–host coupling. For example, Figure 11 ashows a sequence of fluorescence excitation spectra of a singlepentacene molecule in p-terphenyl taken as fast as allowed bythe available SNR, every 3 s. The spectral shifting or hoppingof this molecule from one resonance frequency to anotherfrom scan to scan is clearly evident. Now if the laser frequencyis held fixed near the molecular absorption, then the moleculeappears to blink on and off as it jumps into and out ofresonance (Figure 11 b, at two power levels). Due to the lackof power dependence on the rate, these spontaneous process-es suggested that there are two-level systems available in thehost matrix which can undergo thermally induced transitionseven at these low temperatures. One possible source for thetunneling states in this crystalline system could be discretetorsional librations of the central phenyl ring of the nearby p-

Figure 11. a,b) Spectral diffusion and c) light-induced spectral shifts. From Refs. [48,56,64], respectively. Photos: left: Jim Skinner, right: ThomasBasch¦.


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terphenyl molecules about the molecular axis. The p-terphenyl molecules in a domain wall between two twins ornear lattice defects may have lowered barriers to such central-ring tunneling motions. A theoretical study of the spectraldiffusion trajectories by Jim Skinner and co-workers[60–62]

postulated specific defects that can produce this behavior,attesting to the power of SMS in probing details of the localnanoenvironment and the importance of theoretical insight tofurther understanding. Spectral shifts of single-moleculelineshapes were observed not only for certain crystallinehosts, but also for essentially all polymers studied, and evenfor polycrystalline ShpolÏskii matrices.[63] This is a dramaticexample of the heterogeneity that was uncovered by thesingle-molecule studies.

With my postdoc Thomas Basch¦, light-driven shifts inabsorption frequency were also observed for perylene dopantmolecules in poly(ethylene), in which the rate of the processclearly increased with increases in laser intensity,[64, 65] Fig-ure 11c. This photoswitching effect may be called “spectralhole-burning” by analogy with the earlier hole-burningliterature;[21] however, since only one molecule is in resonancewith the laser, the absorption line simply disappears. Sub-traces (a), (b), and (c) show three successive scans of oneperylene molecule. After trace (c) the laser was tuned intoresonance with the molecule, and at this higher irradiationfluence, eventually the fluorescence signal dropped, that is,the molecule apparently switched off. Trace (d) was thenacquired, which showed that the resonance frequency of themolecule apparently shifted by more than � 1.25 GHz asa result of the light-induced change in the nearby environ-ment. Surprisingly, this effect was reversible for a goodfraction of the molecules: a further scan some minutes later(trace (e)) showed that the molecule returned to the originalabsorption frequency. After trace (g) the molecule wasphotoswitched again and the whole sequence could berepeated many times, enabling us to measure the Poisson

kinetics of this process from the waiting time before a spectralshift.[65]

Several single-molecule systems showed light-inducedshifting behavior at low temperature, for example, terrylenein poly(ethylene),[66] and terrylene in a ShpolÏskii matrix.[67]

Optical modification of single-molecule spectra not onlyprovided a unique window into the photophysics and low-temperature dynamics of the amorphous state, this effectpresaged another area of current interest at room temper-ature: photoswitching of single molecules between emissiveand dark forms is a powerful tool currently being used toachieve super-resolution imaging (see below).

3. Interlude—Why Study Single Molecules?

Before continuing with room-temperature studies, it isuseful to recount some of the key motivations and advantagesof this work. Single-molecule spectroscopy (SMS) allowsexactly one molecule hidden deep within a crystal, polymer,liquid, or cell to be observed via optical excitation of themolecule of interest. This represents detection and spectros-copy at the ultimate sensitivity level of ca. 1.66 × 10¢24 molesof the molecule of interest (1.66 yoctomole), or a quantity ofmoles equal to the inverse of AvogadroÏs number. Detectionof the single molecule must be done in the presence of billionsto trillions of solvent or host molecules. To achieve this, a lightbeam (typically a laser) is used to pump an electronictransition of the one molecule resonant with the opticalwavelength, and it is the interaction of this optical radiationwith the molecule that allows the single molecule to bedetected. Successful experiments must meet the requirementsof 1) guaranteeing that only one molecule is in resonance inthe volume probed by the laser, and 2) providing a signal-to-noise ratio (SNR) for the single-molecule signal that is greaterthan unity for a reasonable averaging time.

Figure 12. Motivations and impact, with selected journal covers from the Moerner lab.


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Why are single-molecule studies now regarded as a criticalpart of modern physical chemistry, chemical physics, andbiophysics (Figure 12)? By removing ensemble averaging, it isnow possible to directly measure distributions of behavior toexplore hidden heterogeneity. This heterogeneity might bestatic, arising from differences in the way in which the singlemolecule interacts with the nearby (complex) environment.Or it might occur in the time domain, arising from the internalstates of one molecule and the transitions among them, andSMS then allows measurement of hidden kinetic pathwaysand the detection of rare short-lived intermediates. Becausetypical single-molecule labels behave like tiny light sourcesroughly 1–2 nm in size and can report on their immediatelocal environment, single-molecule studies provide a newwindow into nanoscale interactions with intrinsic access totime-dependent changes. Fçrster resonant energy transfer(FRET) with single molecules allows detection of conforma-tional changes on the scale of ca. 5 nm.[68] Because the singlemolecule interacts with light primarily via the local electro-magnetic field and the molecular transition dipole moment,enhanced local fields in metallic nanophotonic structures canbe probed.[69, 70] The use of a single molecule as a nm-sizedlight source is one key property used in super-resolutionmicroscopy, described in more detail below after the roomtemperature SMS studies are summarized. Finally, singlemolecules have found commercial application, both in DNAsequencing and in microscopy beyond the diffraction limit.The impact of being able to optically study the smallestindividual component in a complex system is deep and broad.

4. Room Temperature Studies of Single Molecules

Soon after the first low-temperature experiments, studiesbegan of single molecules at room temperature. A selection ofkey milestones are described in Table 1 to the best of myknowledge.

Early steps arose out of the development of “fluorescencecorrelation spectroscopy” (FCS),[72, 73] a large body of workwhich has been extensively reviewed in Refs. [88–90]. Themethod depends upon the fluctuations in emission froma tightly focused spot in solution arising from passage ofmolecules diffusing through a laser beam. Autocorrelationanalysis of the fluorescence provides a window into a varietyof dynamical effects on time scales less than the transit timeon the order of 1–10 ms. The contrast ratio of the autocorre-lation degrades at high concentrations but improves at low,and in 1990 correlation functions were recorded fromconcentrations so low that much less than one molecule wasin the probe volume.[77] The passages of many singlemolecules must be averaged; it is impossible to study onlyone molecule at a time for a long period with FCS.

Also in 1990, the Keller lab at Los Alamos used a carefullydesigned hydrodynamic flow to reduce the volume producinginterfering background signals and directly detected theindividual fluorescence bursts as individual single rhodamine6G molecules passed through the focus.[79] This was a key stepin reducing backgrounds, but there is great value in being ableto watch the same single molecule for extended periods,measuring signal strength, lifetime, polarization, fluctuations,and so on, all as a function of time and with the expresspurpose of directly detecting any heterogeneity from mole-cule to molecule. Hirschfeld reported detection of a singleantibody with 80–100 fluorophores in a short report muchearlier in 1976,[81] but photobleaching and the optical appa-ratus available at the time limited further work.

A key milestone in single-molecule imaging at roomtemperature occurred in 1993, when near-field scanningoptical microscopy (NSOM) was used to lower the pumpedvolume and hence potential interfering backgrounds.[82–84] Itwas subsequently demonstrated that with careful samplepreparation and optimal detection, single molecules could beimaged with far field techniques such as confocal microsco-py,[85] wide-field epifluorescence, and total internal reflection

Table 1: Room temperature milestones of single-molecule detection and imaging.

Solution:Correlationfunctions

Fluorescence Correlation Spectroscopy (FCS): Magde, Elson, Webb (1972);[71–74] Ehrenberg, Rigler (1974);[75] Aragün, Pecora(1976).[76]

Autocorrelation detected from 1 fluorophore or less in the volume: Rigler, Widengren (1990).[77]

Solution:Single bursts

Multichromophore emitter bursts (phycoerythrin): Peck, Stryer, Glaser, Mathies (1989).[78]

Single bursts of fluorescence from 1 fluorophore: Shera, Seitzinger, Davis, Keller, Soper (1990);[79] Nie, Zare (1994).[80]

Solution andsurface

Single antibody with multiple (80–100) labels: Hirschfeld (1976).[81]

Near-FieldNSOM, SNOMImaging

Imaging a single fluorophore: Betzig, Chicester (1993);[82] Ambrose, Goodwin, Martin, Keller (1994);[83] Xie, Dunn (1994).[84]

Confocal imag-ing

Macklin, Trautman, Harris, Brus (1996).[85]

Widefield, singlefluorophoreimaging

In vitro, single myosin on actin: Funatsu, Harada, Tokunaga, Saito, Yanagida (1995).[86]

Cell membrane, single-lipid tracking with super-localization: Schmidt, Schítz, Baumgartner, Gruber, Schindler (1996).[87]


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fluorescence microscopies.[86] Of particular importance forcell biology applications, in 1996 Schmidt et al. explored thediffusion of single labeled lipids on a cell surface.[87] Theexplosion of methods allowing single-molecule detection andimaging has led to a wealth of exciting research in this area,with advances far too numerous to review comprehen-sively,[91–93] and two sets of Nobel Conference Proceedingshave appeared.[94, 95]

4.1. Basics of Single-Molecule Detection and Imaging at RoomTemperature

For concreteness, it is useful at this point to brieflysummarize the basic detection strategy used in modern single-molecule studies at room temperature. Figure 13 illustratessome of the key ideas for the case of cellular imaging, but themethod works for any type of sample as long as theexperiment is designed to strictly reduce background andmaximize the detected emission from the single molecule.Various textbooks may be consulted for additional detail.[96–99]

Typically, organic fluorophore labels (such as TMR (tetra-methyl rhodamine), cyanine dyes like Cy3, Alexa dyes, etc.)or fluorescent protein labels are attached to the biomoleculeof interest, which may be a protein, lipid, sugar, or anoligonucleotide. The pumping light typically excites theenergy levels of the fluorophore as sketched at the upperright, most often an allowed singlet–singlet transition. Vibra-tional relaxation can occur before fluorescence is emitted red-shifted to longer wavelengths, a useful feature that helps inthe detection process—typically long-pass filters are used to

block any scattered pump light. Intersystem crossing canoccur to triplet states, but usually fluorophores are chosen tominimize the time in dark states, except when blinking isrequired. No matter what microscope is used, we can withoutloss of generality think of one diffraction-limited pumpingvolume, which irradiates the sample on a typically transparentsubstrate. Of note is the well-known diffraction limit here:with far-field optics, the focal spot cannot be made smallerthan l/(2 NA) with NA the numerical aperture of themicroscope. In the visible this limit corresponds to about250 nm, and the contrast between the size of the focal spotand the size of the fluorescence labels (a few nm) is dramaticand not to scale. Nevertheless, if the concentration of labeledmolecules is kept low, only one molecule is pumped, and theemitted fluorescence reports on that labeled molecule. Incomparison to the spectral tunability which selects differentmolecules at low temperatures, here brute force dilutionkeeps the molecules separate.

Even without super-resolution methods which resolvedense emitters beyond the diffraction limit (discussed below),many single-molecule studies have been and will continue tobe performed where individual separated molecules areimaged and observed over time. Simply following themotion of the single molecules gives information about thebehavior of the molecules. Figure 14 illustrates severalselected examples of experiments of this type in the Moernerlaboratory from the early 2000s. Figure 14a shows an image ofsingle MHCII (major histocompatibility complexes of type II)proteins anchored in the plasma membrane of a CHO(Chinese hamster ovary) cell. A high affinity antigenicpeptide was labeled with a single fluorophore to light up the

Figure 13. Overview of single-molecule detection and imaging at room temperature. From Ref. [139].


W. E. Moerner



MHCII molecules, and a real-time fluorescence video of themotion of these molecules shows the amazing dance ofMHCIIÏs which occurs on the surface of live cells. Thediffusive properties of the motion and the influence ofcholesterol were studied by my students in collaborationwith Harden McConnell.[100–103]

To give a materials science example, Figure 14b showsa fluorescence image of single molecules of terrylene ina spin-coated crystal of p-terphenyl.[104] Close inspection ofthe image shows that some molecules are small rings, whileothers are unstructured spots. The rings can be understood asthe expected z-oriented dipoles,[105] which are located in well-ordered crystalline regions of the sample. But more informa-tion can be found by watching the images as a function oftime, see the Supporting Information of Ref. [104]. Surpris-ingly, even in this room temperature crystal, the unstructuredspots move around, with highly biased diffusion along roughlyhorizontal and vertical lines in the sample. These singlemolecules are likely moving in the cracks of the sample; thusthe single-molecule motions can be used to visualize thedefects in the crystal.

As a final example of the power of single-moleculetracking, for more than a decade now, the bright and red-shifted emission from single molecules of enhanced yellowfluorescent protein (EYFP) have led to its use as a label forfusions to interacellular proteins in the Moerner lab incollaboration with the laboratory of Lucy Shapiro. Theprimary organism of interest has been Caulobacter crescentus,because cells of this organism display asymmetric division inthe cell cycle: one daughter cell has a flagellum while theother has a stalk with a sticky end. This means that the cellshave a genetic program that causes different groups ofproteins to appear in the two different daughter cells, andunderstanding this process would contribute to the generalproblem of understanding development.[106, 107] The basiceffect arises from spatial patterning of regulatory proteins,which leads to many interesting questions: how do the

proteins actually produce patterns, how do thesepatterns lead to different phenotypes in the daughtercells, and so on. Figure 13c shows what we observedfor single fusions of EYFP to the cytoskeletal proteinMreB in living cells.[108] One population of MreBmolecules were diffusing as expected in the cyto-plasm. However, on a long time scale, time-lapseimaging showed single molecules undergoing cleardirected motion along linear tracks in a circumferen-tial pattern around the edge of the cell. The figureshows tracks for single molecules in different cells,and while this observation was initially thought toinvolve treadmilling of filaments, this behavior islikely associated with MreB molecules interactingwith the cell wall synthesis machinery.[109]

4.2. Toward Super-Resolution: Key Idea #1 isSuperlocalization of Single-Molecule Emitters

We are now in a good position to address super-resolution microscopy with single molecules directly.

As is well-known, biological fluorescence microscopy benefitsfrom a variety of labeling techniques which light up differentstructures in cells, but the price often paid for using visiblelight is the relatively poor spatial resolution compared to X-ray or electron microscopy. Here “resolution” is used in theprecise sense to mean the ability to distinguish two objectsthat are close together. The basic problem briefly mentionedabove is that in conventional far-field microscopes, AbbeÏsfundamental diffraction limit (DL)[110] restricts the resolutionto a value of roughly the optical wavelength l divided by twotimes the numerical aperture (NA) of the imaging system,l/(2 NA). Since the largest values of NA for state-of-the-art,highly corrected immersion microscope objectives are in therange of about 1.3–1.6, the spatial resolution of opticalimaging has been limited to about 200–250 nm for visible lightof 500 nm wavelength.

In fact, the light from single fluorescent molecular labelsabout 1–2 nm in size provides a way around this problem, thatis, a way to provide “super-resolution”, or resolution farbetter than the diffraction limit. (Stimulated emission deple-tion microscopy (STED)[111] and structured illuminationmicroscopy (SIM)[112] are other methods that surpass theDL but do not require single molecules and are discussed byStefan Hell and Eric Betzig elsewhere.) How can singlemolecules help? The sketch in Figure 13 illustrated the typicalimaging problem at room temperature: the single molecule isfar smaller than the focused laser spot, yet, if only onemolecule is pumped, information related to one individualmolecule and its local “nanoenvironment” can be extractedby detecting the photons from that molecule alone.[45] Interms of spatial resolution, however, when the image isformed, the observed “peak” from the single nanoscalesource of light maps out the diffraction-limited point-spreadfunction (PSF) of the microscope, because the molecule isa nanoscale light absorber, far smaller than the size of thePSF. (Rigorously, the single emitting molecule is not strictlya point source, but rather a dipole emitter,[113] but this subtlety

Figure 14. Selected single-molecule imaging and tracking studies at roomtemperature. From Refs. [158] (a), [104] (b), [108] (c). Photos left to right: MarijaVrljic, Stefanie Nishimura, Christopher (Kit) Werley, So Yeon Kim.


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is not important for this discussion.) If many emitters aredecorating a structure, the PSFs overlap to form a blurryimage that is fundamentally “out-of-focus.”

This problem was solved in a direct way by Betzig in2006,[114] by simply preventing all the molecules from emittingat the same time and performing sequential imaging (seeSection 4.4 below). For pedagogical simplicity, I will describethe basic ideas in their simplest form to underscore that theproblem can be solved in a general way. One simply mustfollow two key steps: First, one must be able to acquire theimage of a single molecule and localize its position withprecision much better than the width of the PSF, a processthat may be termed “super-localization.” The second step,active control of the emitting concentration, will then bedescribed in Section 4.4.

Figure 15 illustrates the basic concepts of super-local-ization of single molecules. To state an analogy, anyone canhike up to the top of the cinder cone in the center of CraterLake, Oregon (Figure 15a), and read out the GPS coordinatesof the position of the mountain. This idea is effectivelyapplied to single-molecule emitters: simply by measuring theshape of the PSF, the position of its center can be determined

much more accurately than the PSF width. For example, witha wide-field image of single molecules in Figure 15 b, thediffraction-limited spots are evident. It is essential to spreadout each detected spot on multiple pixels of the camera asshown in Figure 15c. Then, illustrated by a 1D cross-section inFigure 15 d, the various pixels detect different numbers ofphotons according to the shape of the PSF. Formally the PSFis an Airy function, but it may be approximated by a Gaussianfunction for simplicity, especially in the presence of back-ground. The photon numbers detected in the various pixelsprovide samples of the function, which may be fit mathemati-cally. While the width of this fit is still diffraction-limited withwidth w_ , the estimate of the center position c follows a muchnarrower error distribution with standard deviation s, whichis generally called the “localization precision.” The precisionwith which a single molecule can be located by digitizing thePSF depends fundamentally upon the Poisson process ofphoton detection, so the most important variable is the totalnumber of photons detected above background N, witha weaker dependence on the size of the detector pixels andbackground.[115–117] The leading dependence of s is just theAbbe limit divided by the square root of the number of

Figure 15. The central concepts behind super-localization of single-molecule emitters.

Table 2: Early applications of super-localization of single objects in biological imaging.

Find centroidof largefluorescent object

LDL (low-density lipoprotein) particles with many labels on cell surface: Barak, Webb (1982).[118]

Tracking kinesin motor-driven 190 nm bead with few nanometer precision: Gelles, Schnapp, Sheetz (1988).[119]

Find positionof singlefluorophore

Cell membrane, single-lipid tracking to 30 nm precision: Schmidt, Schítz, Baumgartner, Gruber, Schindler (1996).[87]

Single virus particle on HeLa cell to 40 nm precision: Seisenberger,…Br�uchle (2001).[120]


W. E. Moerner



photons detected. This functionalform makes sense, since eachdetected photon is an estimate ofthe molecular position, so for Nmeasurements, the precisionimproves as expected. Super-local-ization means that if 100 photons aredetected, then the precision canapproach 20 nm, and so on. Clearly,then, emitters with the largest num-bers of emitted photons before pho-tobleaching are preferable.

Fitting images to find the centerposition of an object is not a newconcept in science, having beenapplied to experimental data analysisfor some time.[121] In fact, Heisenbergknew in 1930 that the resolutionimprovement improved by one overthe square root of the number ofphotons detected.[122] For concrete-ness, this discussion will be restrictedto biological imaging, and Table 2lists some of the early applicationsto my knowledge. The early cases applied the idea to objectslarger than the diffraction limit such as a LDL particle[118] ora fluorescent bead,[119] and then the localization determined isjust the position of the centroid of the large object. Moreinteresting for the present discussion is the case when a singlefluorophore is emitting, and this type of super-localizationwas first used for tracking single lipids to 30 nm precision bySchmidt et al.[87] A subsequent cellular example addressedsingle virus particle tracking in the process of cellularentry.[120] As selected examples of in vitro studies, digitizationof the PSF for single Cy3 fluorescently-labeled myosinmolecules was used to extract position information down toa few nm by Yildiz et al. ,[123] and a new acronym was proposed(FIONA, for Fluorescence Imaging with One NanometerAccuracy). The knowledge that the same molecule is emittingall the detected photons means that an N-photon correlationis being measured; as long as the photons are independent,the same analysis applies. When more complex photon statescan be used in the future, the situation will change.

4.3. Surprises for Single Fluorescent Protein Molecules: Blinkingand Photocontrol

Another exciting trend in the 1990s was the advent ofgenetically expressed green fluorescent proteins, an area ofgreat importance for molecular and cellular biology whichultimately won the Nobel Prize in Chemistry for OsamuShimomura, Martin Chalfie, and Roger Y. Tsien in 2008.[124]

Indeed, having just left IBM in 1995 for the University ofCalifornia, San Diego, I was able to broaden my interests insingle molecules to include biology and room temperaturestudies. My postdoc, Robert Dickson, and I first worked toachieve partial immobilization of single small organic mole-cules in aqueous environments using the water-filled pores of

poly(acrylamide) gels.[125] Then in 1996, noting the fast-moving events with fluorescent proteins, I had the opportu-nity to obtain samples of a new yellow fluorescent proteinmutant (YFP) from Andy Cubitt in Roger TsienÏs laboratory.As opposed to GFP, which has two absorption bands andundergoes excited state proton transfer from the shorterwavelength band to the longer wavelength form beforeemission,[126] YFP was designed to stabilize the long-wave-length form, and it could be pumped with one of our Ar+ ionlaser lines at 488 nm. Robert Dickson and I then proceeded tosee if we could detect and image single copies of YFP at roomtemperature. Using total internal reflection fluorescence(TIRF) microscopy, Rob was able to record the first imagesof single fluorescent proteins in a gel in 1997[2] as shown inFigure 16 a.

These early experiments also yielded the first example ofa room temperature single-molecule optical switch[2] and thefirst details of the photophysical character of GFP variants onthe single-copy level. The experiments actually utilized twored-shifted GFP variants (S65G/S72A/T203Y denoted“T203Y” and S65G/S72A/T203F denoted “T203F”) whichdiffer only by the presence of a hydroxy group near thechromophore, both of which are quite similar to the widelyused enhanced yellow fluorescent protein EYFP (S65G/V68L/S72A/T203Y). In particular, a fascinating and unex-pected blinking behavior appeared, discernable only on thesingle-molecule level (see the background of Figure 16b fora series of fluorescence images of one molecule for example).This blinking behavior likely results from transformationsbetween at least two states of the chromophore (A and I,Figure 16 d), only one of which (A) is capable of being excitedby the 488 nm pumping laser and producing fluorescence.Additionally, a much longer-lived dark state N was observedupon extended irradiation. Thermally stable in the dark formany minutes, this long-lived dark state was not actually

Figure 16. a) Imaging, b) blinking, and c,d) light-induced photorecovery or switching for singleYFP. Photo: Rob Dickson. From Ref. [2].


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permanently photobleached, rather we found that a little bitof light from a lamp at 405 nm would regenerate the originalfluorescent state as shown in the sequence of images inFigure 16 c. This means that the protein can be used as anemitting label until it enters the long-lived dark state, andthen it can be photo-restored back to the emissive form withthe 405 nm light, a reversal of the apparent photobleaching.When Rob and I observed this blinking and light-inducedrestoration for single copies of YFP, the thought at the timewas the possibility that the photoswitching could be used foroptical storage, and a patent was awarded.

The ability to optically control the emissive states offluorescent proteins quickly expanded, as other researchersaround the world engineered many new photoswitchablefluorescent proteins (such as Kaede,[127] PA-GFP,[128]

EosFP,[129] and DRONPA[130]). These interesting moleculeswith colorful names were to soon play a critical role in thefinal key idea leading to super-resolution microscopy.

4.4. Key Idea #2: Active Control of the Emitting Concentration,and Sequential Imaging

Super-localization works fine when molecules are spa-tially separated, but what can be done when they areoverlapping in the same volume? How can the spatialresolution of such blurry images be improved? It is worthremembering that the low temperature high-resolution spec-troscopy described in Section 2 above provided a potentialclue to this problem: Even within the same diffraction-limitedspot, many different molecules could easily be separatelyselected simply by tuning the laser—the resonance frequencywas a control variable that effectively turned the molecules onand off so that they would not interfere. But we were simplynot thinking of spatial resolution in the early 1990s, becausewe had plenty of spectral resolution! At the same time, in themid-1990s, progress was being made toward general methodsof solving the spatial resolution problem, as summarized inTable 3.

In 1995, after spending years developing near-field opticalimaging at Bell Labs, Eric Betzig wrote a seminal papernoting that a control variable that distinguishes moleculesalong another dimension could be used for super-resolution

microscopy, and he suggested the use of many molecules withdifferent colors, as in the low temperature studies.[131]

Subsequently, in 1998 Antoine van Oijen et al. experimentallydemonstrated this idea directly: they used spectral tunabilityat low temperatures to spatially resolve a set of singlemolecules in three dimensions, with 40 nm lateral and 100 nmaxial resolution, far below the optical diffraction limit.[132,133a]

Of course, biological applications could only become wide-spread if the problem could be solved at room temperature,and researchers continued to try out new ideas to resolveclosely spaced molecules. Multicolor imaging of differentlycolored beads or quantum dots was used to super-resolvea handful of closely-spaced emitters.[133b] Another strategyinvolved using the fluorescence lifetime differences betweenprobes to separate them,[134] demonstrated for two dyesspaced 30 nm apart. Other strategies used naturally occurringphotobleaching—eventually all molecules will bleach exceptone. Adding further to the exploding menagerie of acronyms,this basic idea was demonstrated by Gordon et al. for Cy3labels on DNA[135] (SHRImP, for Single-molecule High-Resolution Imaging with Photobleaching) and by Qu et al.using Cy3-labeled PNA probes on DNA[136] (NALMS, forNAnometer Localized Multiple Single-molecule fluorescencemicroscopy). By separately imaging two fluorophores (Cy3and Cy5) attached to two different calmodulin molecules thatbind to the “legs” of the same single molecule of myosin V,distance measurements accurate to approximately 10 nmwere achieved, and a another acronym was generated[137,140]

(SHREC, for Single-molecule High-REsolution Colocaliza-tion of fluorescent probes). Lidke et al. showed that a certaindegree of super-resolution beyond the diffraction limit couldalso be achieved with the blinking of fluorescent semi-conductor quantum dots.[138]

The stage was now set for several concepts to be puttogether to yield a general method for super-resolutionmicroscopy with single molecules, and key idea #2 isillustrated in Figure 17. A structure has been labeled withmany fluorescent labels as shown on the left, and when all areallowed to emit simultaneously, the blurry image resultsbecause the many PSFs overlap. The key idea is simply to notallow all the molecules to emit at the same time! Let ussuppose that there is some mechanism which allows theemitters to be on part of the time and emitting photons, and

Table 3: Steps toward super-resolution with single-molecule emitters. For acronyms, see Ref. [139]

Key pro-posal

Use some additional control variable to separate DL spots in spatial dimension—spectral tunability suggested: E. Betzig (1995).[131]

Low T Spectral tunability used to achieve 3D super-resolution, 40 nm lateral, 100 nm axial for several single molecules: A. van Oijen, J. Kçhler, J.Schmidt, M. Míller, G. J. Brakenhoff (1998).[132, 133a]

Room T Multicolor imaging to distinguish several fluorescent probes: T. D. Lacoste, X. Michaelet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, S.Weiss (2000).[133b]

Distinguish two dyes by fluorescence lifetime: M. Heilemann, D. P. Herten, R. Heintzmann, C. Cremer, C. Míller, P. Tinnefeld, K. D.Weston, J. Wolfrum, M. Sauer (2002).[134]

Use photobleaching of overlapping fluors: SHRImP: M. P. Gordon, T. Ha, P. R. Selvin (2004);[135] NALMS: X. Qu, D. Wu, L. Mets, N. F.Scherer (2004).[136]

Two differently colored probes: SHREC: L. S. Churchman, Z. Oekten, R. S. Rock, J. F. Dawson, J. F. Spudich (2005).[137]

Blinking of semiconductor quantum dots: K. A. Lidke, B. Rieger, T. M. Jovin, R. Heintzmann (2005).[138]


W. E. Moerner



off, or dark, another part of the time. The experimenter usesthis mechanism to actively control the concentration ofemitting molecules to a very low level such that the PSFs donot overlap. Then using super-localization in one acquiredimage of the molecules, the positions of those are determinedand recorded. Then these molecules are turned off orphotobleached, and another subset is turned on, super-localized, etc. In the end, after a number of sequentialimaging cycles, many locations on the structure have beensampled using the tiny single-molecule “beacons”, and theunderlying image is reconstructed in a pointillist fashion toshow the detail previously hidden beyond the diffraction limiton the right. Effectively, then, the overlapping molecularpositions are determined by time-domain multiplexing.

I first heard about this idea from Eric Betzig and hisprimary collaborator, Harald Hess, in April 2006 at theFrontiers in Live Cell Imaging Conference at the NIH maincampus in Bethesda, Maryland. They used the PA-GFPfluorescent proteins of George Patterson and JenniferLippincott-Schwartz[128] and other photoswitchable fluores-cent proteins as an active control mechanism, terming themethod PALM (for PhotoActivated Localization Microsco-py).[114] Light-induced photoactivation of GFP mutant fusionsis used to randomly turn on only a few single molecules ata time in fixed cell sections or fixed cells. In their tour de forceexperiment, individual PSFs were recorded in detail to findtheir positions to approximately 20 nm, then were photo-bleached so that others could be turned on, and so on untilmany thousands of PSF positions were determined, anda super-resolution reconstruction was produced.

Very quickly after the NIH meeting, a flood of researchersdemonstrated super-resolution imaging with single moleculesusing additional active control mechanisms and additionalacronyms. The laboratory of Xiaowei Zhuang utilized con-trolled photoswitching of small molecule fluorophores forsuper-resolution demonstrations[141] (STORM, for STochasticOptical Reconstruction Microscopy). Their original methodused a Cy3-Cy5 emitter pair in close proximity that shows

a novel property: restoration of Cy5Ïs photo-bleached emission can be achieved by brief pump-ing of the Cy3 molecule. In this way, the emissionfrom a single Cy5 on DNA or an antibody is turnedon by pumping Cy3 and off by photobleaching,again and again, in order to measure its positionaccurately multiple times. After many such deter-minations, the localization accuracy can approachapproximately 20 nm precision, and labeled anti-bodies (labeled with > 1Cy3, ! 1Cy5) were usedto localize RecA proteins bound to DNA. SamuelHess et al. published an approach similar toBetzigÏs with an acronym termed F-PALM (Fluo-rescence PhotoActivation Localization Microsco-py),[142] which also utilized a photoactivatable GFPwith PSF localization to obtain superresolution.Also in 2006, an alternative approach was reportedby the laboratory of Robin Hochstrasser based onaccumulated binding of diffusible probes, whichare quenched in solution yet de-quench in closeproximity of the surface of the object to be

imaged[143] (termed PAINT, for Points Accumulation forImaging in Nanoscale Topography). The method relies uponthe photophysical behavior of certain molecules that light upwhen bound or constrained, and they demonstrated the ideawith the twisted intermolecular charge transfer (TICT) stateof Nile Red.[144] PAINT has advantages that the object to beimaged need not be labeled and that many individualfluorophores are used for the imaging, thus relaxing therequirement on the total number of photons detected fromeach single molecule.

Other active control mechanisms quickly appeared suchas dSTORM[145] (direct STORM), GSDIM (Ground-StateDepletion with Intermittent Return),[146] blinking as inBLINK microscopy,[147] SPDM (Spectral Precision Determi-nation Microscopy),[148] and the list goes on. In 2008, JulieBiteen in my laboratory used the EYFP photorecoverymechanism described above to perform super-resolutionimaging in bacteria,[149] but since we did not create a newacronym for this, the work did not receive as much attention.Therefore, to jokingly add a new acronym to the field that ismechanism-independent, my lab informally uses the acronymSMACM, which stands for Single-Molecule Active ControlMicroscopy. In any case, the key underlying idea is verygeneral, and PALM led the way. There are photochemicalmethods for single-fluorophore turn-on[150] and even enzy-matic methods for turn-on which may be controlled by theconcentration of substrate and the enzymatic rate.[151] Theexperimenter must actively choose some method to controlthe emitting concentration. Of course, the imaging is stilltime-sequential, thus this approach is best for quasi-staticstructures or fixed cells, but significant progress has beenmade in increasing the imaging speed.[152] Selected reviewsmay be consulted for additional detail of modern challengesand progress.[153–161]

Figure 17. Active control of emitting concentration leads to super-resolution micros-copy. From Ref. [155].


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4.5. Super-Resolution Microscopy Applications andDevelopments from the Moerner Lab

Since the early 2000s, my laboratory in the StanfordChemistry Department has been in a fruitful collaborationwith the microbiology and developmental biology laboratoryof Lucy Shapiro to use advanced single-molecule imaging toexplore regulatory protein localization patterns in a particu-larly interesting bacterium, Caulobacter crescentus. Sincebacteria are very small, only a couple of microns long andsubmicron in diameter, the size of the entire organism is nearthe optical diffraction limit and super-resolution microscopy

can be used to great advantage. Thus, as mentioned in the lastsection, in 2007 we began single-molecule super-resolutionimaging in bacteria, and took advantage of the photoinducedrecovery and/or blinking of single EYFP we discovered in1997[2] as an active-control mechanism. Figure 18 illustrateshow the raw data actually appear: Figure 18 a shows a whitelight transmission image of a field of cells, and Figure 18bshows a single fluorescence frame after initial bleachdown.From many 10–50 ms frames such as these, super-localizationis performed to extract single-molecule localizations, andsuper-resolution reconstructions can be generated.

Figure 19 illustrates some of the super-resolution imagesfrom three of our Caulobacter studies in recent years. Theupper row shows what would be observed with diffraction-limited conventional fluorescence imaging, and the lower rowshows SMACM super-resolution images of the same cells. Ineach column, a different target protein has been fused toEYFP. Column 1 shows imaging results from my postdoc atthe time, Julie Biteen, on the MreB cytoskeletal protein whichappears to form a quasi-helical structure.[149] (Later worknoted that the helical shape is likely an artifact of thefluorescent protein construct that was used.[162] Super-reso-lution naturally provides higher resolution that allows sucheffects to be observed, so additional care must now be takento guard against labeling perturbation and to developimproved labels.) Column 2 shows ParA protein resultsgenerated by a collaboration between Jerod Ptacin fromLucyÏs lab and my postdoc, Steven Lee.[163] Involved in the

Figure 18. Raw data showing blinking of single EYFP fusions toa target protein in Caulobacter bacteria. a) White light transmissionimage. b) Single 10 ms frame of fluorescence image, 5 mm scale bar.

Figure 19. Super-resolution imaging of three different proteins in Caulobacter: MreB,[149] ParA,[163] HU2.[164] Photos left to right: Julie Biteen,Stephen Lee, Mike Thompson.


W. E. Moerner



process of chromosome segregation, ParA localized ina narrow linear structure running along the axis of the cell,which recedes during the translocation of the chromosomalorigin from the old pole to the new pole. Finally, column 3shows fixed-cell data for the nucleoid binding protein HU2,from work by Steven Lee and graduate student MikeThompson.[164] Because HU2 binds nonspecifically to manylocations on the chromosome, the localizations here provideuseful information about the DNA distribution inside the cellwhich could be analyzed with spatial point statistics. Overall,these images show how important super-resolution imaging isin providing detail that could not be observed before, andsuper-resolution imaging is widely used for bacteria atpresent.[165–167]

Of course, super-resolution imaging in eukaryotic cells isalso a major area of current interest. In Figure 20, I includeone example from my lab utilizing a novel method ofachieving active control, which might be called target-specificPAINT, enabled by a collaboration with the syntheticchemistry laboratory of Justin Du Bois at Stanford.[168]

Alison Ondrus, a postdoc in the Du Bois lab, was able tosynthesize the potent neurotoxin molecule shown inFigure 20, saxitoxin (STX), with a covalently attached fluo-rescent label such as Cy5. Given this fluorescent ligand whichbinds to and blocks voltage-gated sodium (NaV) channels, itwas then possible for my graduate student, Hsiao-lu Lee, anda visiting scholar, Shigeki Iwanaga, to grow PC12 cells ona coverslip surface, induce them to differentiate into neural-like cells, and then simply provide the STX-Cy5 to thesolution above the cells. The ligands in solution are not easilyimaged due to their fast motion. Diffusion brings the STX-Cy5 to the cell surface where the molecule binds to NaV

channels and provides a bright fluorescent spot for super-

localization. The label then photobleaches and dissociatesfrom the cell, allowing new ligands to bind. By recordinga fluorescent movie, many single-molecule localizations couldbe continuously recorded and grouped to form a super-resolution reconstruction of the locations of the channels onthe cell membrane. Figure 20 shows data recorded fromaxonal-like projections, where the panels on the rightcompare diffraction-limited and super-resolution reconstruc-tions. By grouping all localizations within a 6.25 s interval, thesequence of images on the left shows how the cell changesover time, with various sub-diffraction neuritic extensionsgrowing and retracting. It was also possible to record time-dependent images on a time scale of 500 ms by sliding boxcaraveraging (see Supporting Information of Ref. [168].) Thus,with this method, a reasonable degree of time-dependentbehavior can be observed, well beyond the diffraction limit.

Another recent application of PALM/SMACM to eukary-otic cells involved imaging of Huntingtin (Htt) proteinaggregate structures in cells. The Htt protein leads to theneurodegenerative HuntingtonÏs disease when the poly(glut-amine) repeat sequence is expanded. Super-resolution imagesof the aggregate structures were imaged in vitro by mygraduate student Whitney Duim.[169,170] In a collaborationwith the laboratory of Judith Frydman at Stanford, mypostdoc Steffen Sahl and graduate student Lucien Weiss grewneuronal model PC12m cells transfected with the mutantform of the Htt protein exon 1 fused to EYFP and imaged thefluorescence from fixed and live cells at various time pointspost-transfection.[171] Critical to success of these experimentswas targeted photobleaching of the extremely bright inclusionbody (IB) before single-molecule imaging of the blinkingEYFP. In this way, it was possible to observe tiny aggregatespecies in the cell body as shown in Figure 21 a, with reversed-

Figure 20. Example of super-resolution cellular imaging using a fluorescent saxitoxin ligand binding to ion channels on the cell surface. Bar in leftsequence: 5 mm. From Ref. [168]. Photos: Hsiao-lu Lee, Shigeki Iwanaga.


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Figure 21. Super-resolution imaging of Htt fibrillary aggregates in cells. a) Cell body, with 500 nm scale bar in the lower images. b) Axonalprocesses. Scale bar 5 mm in upper images, 500 nm lower. Photos: Steffen Sahl, Lucien Weiss. From Ref. [171].

Figure 22. New photoswitchable fluorophores (left) and 3D imaging strategies (right). Photo: Marissa Lee. From Ref. [172].


W. E. Moerner



contrast super-resolution reconstructions showing that theseare small fibrillary structures. In axonal-like projections fromthe cells (Figure 21b), various small aggregate species are alsoobserved with super-resolution detail. It is not fully known atthe present time whether or not these small aggregate speciesare themselves toxic or the product of cellular processing toremove them, but being able to image and quantify suchstructures is an important start toward understanding themechanism of the disease, and this method is being applied toother neurodegenerative disorders.

To end this very brief summary of super-resolutionimaging with single molecules, I want to mention a coupleof the outstanding challenges and current directions ofdevelopment, using the illustrations in Figure 22. One areais the need for better fluorophores, specifically molecules withthe ability to be turned on (and off) at will, with more emittedphotons than are available from fluorescent proteins, forexample. Small organic molecules generally offer ten timesmore total emitted photons than fluorescent proteins andcould be less perturbative, so combining such molecules witha photochemical or photophysical mechanism for turn-onwould be preferable. (Of course, it is also necessary to targetsuch molecules to appropriate biomolecules, and much effortis going on in this area, too.) The left side of Figure 22 showsa photoswitchable rhodamine spirolactam which has beenmodified by Prabin Rai in the laboratory of my syntheticcollaborator, Robert Twieg at Kent State University, toundergo photoinduced turn-on by opening of the lactam ringwith blue rather than ultraviolet light.[172] Using an N-hydroxysuccinimide derivative, my graduate student MarissaLee covalently attached these molecules to the surface of liveCaulobacter cells and then recorded super-resolution imagesof the cell surface. The images at the bottom show that thismethod produces excellent reconstructions with many local-izations, and the sub-diffraction-sized bacterial stalks ofvarying lengths are easily observedand quantified.

Another area of intense currentinterest is the extension of super-resolution microscopy beyond twospatial transverse dimensions tothree dimensions, x, y, and z. Whilesome researchers have pursued astig-matic imaging,[173] multi-plane imag-ing,[174, 175] or other approaches, mylaboratory has concentrated onadvanced methods of pupil-planeoptical Fourier processing[176] toencode the z-position of single mol-ecules in the shape of the PSF itself.Our first step in this area was incollaboration with Rafael Piestun ofthe University of Colorado to dem-onstrate that the double-helix pointspread function (DH-PSF) can beused for single-molecule microsco-py.[177] The right side of Figure 22,upper panel, shows that the DH-PSFoperation converts the usual single

spot from a single molecule into two spots that revolve aroundone another depending upon the z-position of the molecule inthe sample. The angle of the line between the two spotsencodes the z-position simultaneously for all molecules in theframe over a 2 mm depth of field. This approach has superiorFisher information and thus better localization precision thanthat of other approaches,[178] and we have used the DH-PSF intwo colors to co-image two different fluorescent proteinfusions in Caulobacter.[159,179] The lower half of Figure 22shows the application of the DH-PSF method to 3D surfaceimaging of Caulobacter labeled by the rhodamine spirolac-tam.[172] Much remains to be done, as new point-spreadfunction designs continue to appear,[180–182] with the continu-ing goal to extract the maximum information from each tinysingle-molecule emitter in the most efficient fashion.

5. Concluding Remarks and Acknowledgements

In this contribution, the early steps leading to the firstsingle-molecule detection and spectroscopy,[1,27] were de-scribed. The low temperature imaging experiments in theearly 1990s yielded many novel physical effects, such asspectral diffusion and light-activated switching which havereappeared in the later room temperature studies in different,but related forms. At room temperature, the surprising single-molecule blinking and photoswitching for single GFP mole-cules provided a pathway to the active control that wasneeded for PALM super-resolution microscopy and itsrelatives. Today, super-resolution microscopy is a powerfulapplication of single molecules that has broad impact acrossmany fields of science (Figure 23), and new and amazingdiscoveries continue, such as the observation of actin bands inaxons.[183] All of this has occurred due not only to my efforts,but also due in major part to the clever and insightful research

Figure 23. Impact of single-molecule spectroscopy and imaging, with selected examples. Multicolor3D image of intracellular proteins and cell surface courtesy Matthew Lew (pictured); see Ref. [179].


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performed by many researchers around the world toonumerous to mention here. Beyond super-resolution micros-copy, just observing single molecules and their behaviorscontinues to lead to tantalizing scientific advances, whetherthis is simply tracking single-molecule motions,[184] or infer-ring biomolecular interactions and conformations withFRET[185, 186] or extracting photodynamics from trappedsingle molecules,[187,188] or determining enzymatic mecha-nisms.[189] The future of single-molecule spectroscopy andsuper-resolution imaging is very bright.

I have been extremely fortunate throughout the entireperiod of my research career to have had the privilege ofworking with a team of brilliant and exceptional students andpostdoctoral researchers. The Moerner lab alumni are listed inFigure 24, and I warmly thank all of them for their hard workand insights. I am also extremely grateful to my currentstudents and postdocs, pictured near the Rodin SculptureGarden on Halloween in Figure 25, along with our “NoEnsemble Averaging” logo from Sam Lord. These talentedscientists are continuing to push the field of single-moleculespectroscopy, trapping, imaging, and super-resolution into thefuture. The figure explains why we like to refer to onemolecule as a “guacamole” of material! My education andresearch ever since my college years have benefited fromnumerous wonderful collaborators and colleagues listed inFigure 26, and I have truly enjoyed being a student of many ofthem. I am sure that some have been left out for which Iapologize. Of course, I owe a special personal and professionaldebt to my spectacular mentors, the institutions who havehosted me, and the various funding agencies, administrators,and staff that have supported my work listed on Figure 27.Finally, on Figure 28, I sincerely and deeply thank my familyand my close personal friends who have listened to andcounseled me through many challenges over the years. Myparents sacrificed a great deal for me and provided continuinglove and support to me throughout their lives, and I havethoroughly enjoyed the well-wishes and encouragement fromall members of my extended family. My son Daniel, aninquisitive and deep thinker, always listens and continues toamaze me, and he provides a continuing inspiration for thefuture. My wife, Sharon, has been an indispensable source oflove, companionship, patience, and encouragement to methroughout our marriage, and I cannot thank her enough.

Figure 24. Moerner lab alumni.

Figure 25. The current guacamole team.

Figure 26. My collaborators/colleagues.


W. E. Moerner



How to cite: Angew. Chem. Int. Ed. 2015, 54, 8067–8093Angew. Chem. 2015, 127, 8182–8210

[1] “Optical detection and spectroscopy of single molecules ina solid”: W. E. Moerner, L. Kador, Phys. Rev. Lett. 1989, 62,2535 – 2538.

[2] “On/Off blinking and switching behavior of single molecules ofgreen fluorescent protein”: R. M. Dickson, A. B. Cubitt, R. Y.Tsien, W. E. Moerner, Nature 1997, 388, 355 – 358.

[3] “Laser spectroscopy of trapped atomic ions”: W. M. Itano, J. C.Bergquist, D. J. Wineland, Science 1987, 237, 612.

[4] “Laser experiments with single atoms as A test of basicphysics”: F. Diedrich, J. Krause, G. Rempe, M. O. Scully, H.Walther, IEEE J. Quantum Electron. 1988, 24, 1314.

[5] “Experiments with an isolated subatomic particle at rest”: H.Dehmelt, Rev. Mod. Phys. 1990, 62, 525 – 530.

[6] “Scanning tunneling microscopy-from birth to adolescence”: G.Binnig, H. Rohrer, Rev. Mod. Phys. 1987, 59, 615.

[7] “Atomic force microscope”: G. Binnig, C. F. Quate, C. Gerber,Phys. Rev. Lett. 1986, 56, 930 – 933.

[8] “Single-channel currents recorded from membrane of dener-vated frog muscle fibres”: E. Neher, B. Sakmann, Nature 1976,260, 799 – 802.

[9] “Are there quantum jumps? part II”: E. R. J. A. Schrçdinger,Br. J. Phil. Sci. 1952, 3, 233 – 242.

[10] a) M. Pope, C. E. Swenberg, Electronic Processes in OrganicCrystals and Polymers, Oxford Univ. Press, London, 1999 ;b) “Absorption, excitation, and emission spectroscopy of terry-lene in p-terphenyl: Bulk measurements and single molecule

studies”: S. Kummer, F. Kulzer, R. Kettner, T. Basch¦, C. Tietz,C. Glowatz, C. Kryschi, J. Chem. Phys. 1997, 107, 7673 – 7684.

[11] “Radiationless relaxation and optical dephasing of moleculesexcited by wide- and narrow-band lasers. II. pentacene in low-temperature mixed crystals”: T. E. Orlowski, A. H. Zewail, J.Chem. Phys. 1979, 70, 1390 – 1426.

[12] “Shapes of inhomogeneously broadened resonance lines insolids”: A. M. Stoneham, Rev. Mod. Phys. 1969, 41, 82.

[13] “The effect of fine structure appearance in laser-excitedfluorescence spectra of organic compounds in solid solutions”:R. I. Personov, E. I. AlÏShits, L. A. Bykovskaya, Opt. Commun.1972, 6, 169 – 173.

[14] “High-resolution spectroscopy of organic molecules in solids:From fluorescence line narrowing and hole burning to singlemolecule spectroscopy”: M. Orrit, J. Bernard, R. I. Personov, J.Phys. Chem. 1993, 97, 10256 – 10268.

[15] “Fluorescence transient and optical free induction decayspectroscopy of pentacene in mixed crystals at 2 K. determi-nation of intersystem crossing and internal conversion rates”:H. de Vries, D. A. Wiersma, J. Chem. Phys. 1979, 70, 5807.

[16] “Coherent optical transient studies of dephasing and relaxationin electronic transitions of large molecules in the condensedphase”: D. A. Wiersma, Adv. Chem. Phys. 1981, 47, 421.

[17] “Dynamics in low temperature glasses: Theory and experi-ments on optical dephasing, spectral diffusion, and hydrogentunneling”: M. Berg, C. A. Walsh, L. R. Narasimhan, K. A.Littau, M. D. Fayer, J. Chem. Phys. 1988, 88, 1564 – 1587.

[18] “Hole burning in the contour of a pure electronic line ina ShpolÏskii system”: A. A. Gorokhovskii, R. K. Kaarli, L. A.Rebane, JETP Lett. 1974, 20, 216.

[19] “Stable ”gap“ in absorption spectra of solid solutions of organicmolecules by laser irradiation”: B. M. Kharlamov, R. I. Per-sonov, L. A. Bykovskaya, Opt. Commun. 1974, 12, 191 – 193.

[20] “Molecular electronics for frequency domain optical storage:Persistent spectral hole-burning – a review”: W. E. Moerner, J.Mol. Electron. 1985, 1, 55 – 71.

[21] W. E. Moerner, Persistent Spectral Hole-Burning: Science andApplications (Top. Curr. Phys. 44), Springer, Berlin, 1988.

[22] “Photochemical hole burning: A spectroscopic study of relax-ation processes in polymers and glasses”: J. Friedrich, D.Haarer, Angew. Chem. Int. Ed. Engl. 1984, 23, 113 – 140;Angew. Chem. 1984, 96, 96 – 123.

[23] “Mechanisms of non-photochemical hole-burning in organicglasses”: J. M. Hayes, G. J. Small, Chem. Phys. Lett. 1978, 54,435 – 438.

[24] “Non-photochemical hole burning and impurity site relaxationprocesses in organic glasses”: J. M. Hayes, G. J. Small, Chem.Phys. 1978, 27, 151 – 157.

[25] “From spectral holeburning memory to spatial-spectral micro-wave signal processing”: W. R. Babbitt, Z. W. Barber, S. H.Bekker, M. D. Chase, C. Harrington, K. D. Merkel, R. K.Mohan, T. Sharpe, C. R. Stiffler, A. S. Traxinger, A. J. Woidtke,Laser Phys. 2014, 24, 094002.

[26] “Can single-photon processes provide useful materials forfrequency-domain optical storage?”: W. E. Moerner, M. D.Levenson, J. Opt. Soc. Am. B 1985, 2, 915 – 924.

[27] “Statistical fine structure in inhomogeneously broadenedabsorption lines”: W. E. Moerner, T. P. Carter, Phys. Rev.Lett. 1987, 59, 2705.

[28] “Statistical fine structure in the inhomogeneously broadenedelectronic origin of pentacene in p-terphenyl”: T. P. Carter, M.Manavi, W. E. Moerner, J. Chem. Phys. 1988, 89, 1768.

[29] “Frequency-modulation spectroscopy: A new method formeasuring weak absorptions and dispersions”: G. C. Bjorklund,Opt. Lett. 1980, 5, 15.

[30] “Pseudo-stark effect and FM/Stark double-modulation spec-troscopy for the detection of statistical fine structure in

Figure 28. My friends and family.

Figure 27. My mentors, institutions, and funding sources.


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http://dx.doi.org/10.1103/PhysRevLett.62.2535


http://dx.doi.org/10.1126/science.237.4815.612

http://dx.doi.org/10.1109/3.968

http://dx.doi.org/10.1103/RevModPhys.62.525



http://dx.doi.org/10.1038/260799a0

http://dx.doi.org/10.1038/260799a0

http://dx.doi.org/10.1063/1.475107

http://dx.doi.org/10.1063/1.437579

http://dx.doi.org/10.1063/1.437579


http://dx.doi.org/10.1016/0030-4018(72)90220-9

http://dx.doi.org/10.1016/0030-4018(72)90220-9

http://dx.doi.org/10.1021/j100142a003

http://dx.doi.org/10.1021/j100142a003

http://dx.doi.org/10.1063/1.437411

http://dx.doi.org/10.1063/1.454136

http://dx.doi.org/10.1016/0030-4018(74)90388-5



http://dx.doi.org/10.1016/0009-2614(78)85255-5

http://dx.doi.org/10.1016/0009-2614(78)85255-5

http://dx.doi.org/10.1016/0301-0104(78)85169-6

http://dx.doi.org/10.1016/0301-0104(78)85169-6

http://dx.doi.org/10.1088/1054-660X/24/9/094002

http://dx.doi.org/10.1364/JOSAB.2.000915



http://dx.doi.org/10.1063/1.455123

http://dx.doi.org/10.1364/OL.5.000015


alexandrite”: T. P. Carter, D. E. Horne, W. E. Moerner, Chem.Phys. Lett. 1988, 151, 102.

[31] “Frequency modulation (FM) spectroscopy”: G. C. Bjorklund,M. D. Levenson, W. Lenth, C. Ortiz, Appl. Phys. B 1983, 32, 145.

[32] “Optical detection and probing of single dopant molecules ofpentacene in a p-terphenyl host crystal by means of absorptionspectroscopy”: L. Kador, D. E. Horne, W. E. Moerner, J. Phys.Chem. 1990, 94, 1237 – 1248.

[33] “Residual amplitude modulation in laser electro-optic phasemodulation”: E. A. Whittaker, M. Gehrtz, G. C. Bjorklund, J.Opt. Soc. Am. B 1985, 2, 1320.

[34] “Absorption spectroscopy on single molecules in solids”: L.Kador, T. Latychevskaia, A. Renn, U. P. Wild, J. Chem. Phys.1999, 111, 8755 – 8758.

[35] “Single pentacene molecules detected by fluorescence excita-tion in a p-terphenyl crystal”: M. Orrit, J. Bernard, Phys. Rev.Lett. 1990, 65, 2716 – 2719.

[36] “Spectroscopy of K2 using laser-induced fluorescence”: W. J.Tango, J. K. Link, R. N. Zare, J. Chem. Phys. 1968, 49, 4264 –4268.

[37] “Stark effect on single molecules in a polymer matrix”: M.Orrit, J. Bernard, A. Zumbusch, R. I. Personov, Chem. Phys.Lett. 1992, 196, 595.

[38] “Probing individual two-level systems in a polymer by corre-lation of single-molecule fluorescence”: A. Zumbusch, L.Fleury, R. Brown, J. Bernard, M. Orrit, Phys. Rev. Lett. 1993,70, 3584 – 3587.

[39] “Single molecule polarization spectroscopy: Pentacene in p-terphenyl”: F. Gîttler, M. Croci, A. Renn, U. P. Wild, Chem.Phys. 1996, 211, 421 – 430.

[40] Single-Molecule Optical Detection, Imaging, and Spectroscopy(Eds.: T. Basch¦, W. E. Moerner, M. Orrit, U. P. Wild), VerlagChemie, Weinheim, 1997.

[41] “Optical spectroscopy of single impurity molecules in solids”:W. E. Moerner, T. Basch¦, Angew. Chem. Int. Ed. Engl. 1993,32, 457; Angew. Chem. 1993, 105, 537.

[42] “Single-molecule spectroscopy and quantum optics in solids”:W. E. Moerner, R. M. Dickson, D. J. Norris, Adv. At. Mol. Opt.Phys. 1998, 38, 193 – 236.

[43] “Structure and dynamics in solids as probed by opticalspectroscopy”: J. L. Skinner, W. E. Moerner, J. Phys. Chem.1996, 100, 13251 – 13262.

[44] “High-resolution optical spectroscopy of single molecules insolids”: W. E. Moerner, Acc. Chem. Res. 1996, 29, 563.

[45] “Examining nanoenvironments in solids on the scale of a single,isolated molecule”: W. E. Moerner, Science 1994, 265, 46 – 53.

[46] “Optical spectroscopy of single molecules in solids”: M. Orrit, J.Bernard, R. Brown, B. Lounis, Prog. Opt. 1996, 35, 61 – 144.

[47] “Single-molecule spectroscopy”: T. Plakhotnik, E. A. Donley,U. P. Wild, Annu. Rev. Phys. Chem. 1997, 48, 181 – 212.

[48] “Fluorescence spectroscopy and spectral diffusion of singleimpurity molecules in a crystal”: W. P. Ambrose, W. E.Moerner, Nature 1991, 349, 225 – 227.

[49] “Detection and spectroscopy of single pentacene molecules ina p-terphenyl crystal by means of fluorescence excitation”:W. P. Ambrose, T. Basch¦, W. E. Moerner, J. Chem. Phys. 1991,95, 7150 – 7163.

[50] “Photon antibunching in the fluorescence of a single dyemolecule trapped in a solid”: T. Basch¦, W. E. Moerner, M.Orrit, H. Talon, Phys. Rev. Lett. 1992, 69, 1516 – 1519.

[51] “Dispersed fluorescence spectra of single molecules of penta-cene in p-terphenyl”: P. Tch¦nio, A. B. Myers, W. E. Moerner, J.Phys. Chem. 1993, 97, 2491.

[52] “Vibrational analysis of dispersed fluorescence from singlemolecules of terrylene in polyethylene”: P. Tch¦nio, A. B.Myers, W. E. Moerner, Chem. Phys. Lett. 1993, 213, 325.

[53] “Vibronic spectroscopy of individual molecules in solids”: A. B.Myers, P. Tch¦nio, M. Zgierski, W. E. Moerner, J. Phys. Chem.1994, 98, 10377.

[54] “Magnetic resonance of a single molecular spin”: J. Kçhler,J. A. J. M. Disselhorst, M. C. J. M. Donckers, E. J. J. Groenen, J.Schmidt, W. E. Moerner, Nature 1993, 363, 242 – 244.

[55] “Near-field optical spectroscopy of individual molecules insolids”: W. E. Moerner, T. Plakhotnik, T. Irngartinger, U. P.Wild, D. Pohl, B. Hecht, Phys. Rev. Lett. 1994, 73, 2764.

[56] “Comment on single pentacene molecules detected by fluores-cence excitation in a p-terphenyl crystal”: W. E. Moerner, W. P.Ambrose, Phys. Rev. Lett. 1991, 66, 1376.

[57] “Intersystem crossing from singlet states of molecular dimersand monomers in mixed molecular crystals: Picosecond stimu-lated photon echo experiments”: F. G. Patterson, H. W. H. Lee,W. L. Wilson, M. D. Fayer, Chem. Phys. 1984, 84, 51.

[58] “Fluorescence microscopy of single molecules”: F. Gîttler, T.Irngartinger, T. Plakhotnik, A. Renn, U. P. Wild, Chem. Phys.Lett. 1994, 217, 393.

[59] Structural relaxation processes in polymers and glasses asstudied by high resolution optical spectroscopy: J. Friedrich, D.Haarer, in Optical Spectroscopy of Glasses (Ed.: I. Zschokke),Reidel, Dordrecht, 1986, p. 149.

[60] “Spectral diffusion of single molecule fluorescence: A probe oflow-frequency localized excitations in disordered crystals”:P. D. Reilly, J. L. Skinner, Phys. Rev. Lett. 1993, 71, 4257 – 4260.

[61] “Spectral diffusion of individual pentacene molecules in p-terphenyl crystal: Stochastic theoretical model and analysis ofexperimental data”: P. D. Reilly, J. L. Skinner, J. Chem. Phys.1995, 102, 1540.

[62] “Theory of single-molecule optical line-shape distributions inlow-temperature glasses”: E. Geva, J. L. Skinner, J. Phys.Chem. B 1997, 101, 8920 – 8932.

[63] “Single-molecule spectroscopy in Shpol’skii matrices”: T.Plakhotnik, W. E. Moerner, T. Irngartinger, U. P. Wild,Chimia 1994, 48, 31.

[64] “Optical modification of a single impurity molecule in a solid”:T. Basch¦, W. E. Moerner, Nature 1992, 355, 335 – 337.

[65] “Optical spectra and kinetics of single impurity molecules ina polymer: Spectral diffusion and persistent spectral hole-burning”: T. Basch¦, W. P. Ambrose, W. E. Moerner, J. Opt.Soc. Am. B 1992, 9, 829.

[66] “Optical studies of single terrylene molecules in polyethylene”:P. Tch¦nio, A. B. Myers, W. E. Moerner, J. Lumin. 1993, 56, 1.

[67] “Optical probing of single molecules of terrylene in a shpolskiimatrix – A two-state single-molecule switch”: W. E. Moerner,T. Plakhotnik, T. Irngartinger, M. Croci, V. Palm, U. P. Wild, J.Phys. Chem. 1994, 98, 7382 – 7389.

[68] “Probing the interaction between two single molecules: Fluo-rescence resonance energy transfer between a single donor anda single acceptor”: T. Ha, T. Enderle, D. F. Ogletree, D. S.Chemla, P. R. Selvin, S. Weiss, Proc. Natl. Acad. Sci. USA 1996,93, 6264 – 6268.

[69] Nanophotonics and single molecules: W. E. Moerner, P. J.Schuck, D. P. Fromm, A. Kinkhabwala, S. J. Lord, S. Y.Nishimura, K. A. Willets, A. Sundaramurthy, G. S. Kino, M.He, Z. Lu, R. J. Twieg, in Single Molecules and Nanotechnology(Eds.: R. Rigler, H. Vogel), Springer, Berlin, 2008, pp. 1 – 23.

[70] “Large single-molecule fluorescence enhancements producedby a gold bowtie nanoantenna”: A. Kinkhabwala, Z. Yu, S. Fan,Y. Avlasevich, K. Mullen, W. E. Moerner, Nat. Photonics 2009,3, 654.

[71] “Thermodynamic fluctuations in a reacting system – measure-ment by fluorescence correlation spectroscopy”: D. Magde, E.Elson, W. W. Webb, Phys. Rev. Lett. 1972, 29, 705.

[72] “Fluorescence correlation spectroscopy. I. conceptual basis andtheory”: E. L. Elson, D. Magde, Biopolymers 1974, 13, 1 – 27.


W. E. Moerner


http://dx.doi.org/10.1016/0009-2614(88)80078-2

http://dx.doi.org/10.1016/0009-2614(88)80078-2

http://dx.doi.org/10.1007/BF00688820

http://dx.doi.org/10.1021/j100367a011

http://dx.doi.org/10.1021/j100367a011



http://dx.doi.org/10.1063/1.480222

http://dx.doi.org/10.1063/1.480222



http://dx.doi.org/10.1063/1.1669869

http://dx.doi.org/10.1063/1.1669869

http://dx.doi.org/10.1016/0009-2614(92)86000-8

http://dx.doi.org/10.1016/0009-2614(92)86000-8



http://dx.doi.org/10.1016/0301-0104(96)00250-9

http://dx.doi.org/10.1016/0301-0104(96)00250-9




http://dx.doi.org/10.1021/jp9601328

http://dx.doi.org/10.1021/jp9601328

http://dx.doi.org/10.1021/ar950245u


http://dx.doi.org/10.1146/annurev.physchem.48.1.181

http://dx.doi.org/10.1038/349225a0

http://dx.doi.org/10.1063/1.461392

http://dx.doi.org/10.1063/1.461392


http://dx.doi.org/10.1016/0009-2614(93)85140-J

http://dx.doi.org/10.1021/j100092a001

http://dx.doi.org/10.1021/j100092a001

http://dx.doi.org/10.1038/363242a0



http://dx.doi.org/10.1016/0301-0104(84)80005-1

http://dx.doi.org/10.1016/0009-2614(94)87003-9

http://dx.doi.org/10.1016/0009-2614(94)87003-9


http://dx.doi.org/10.1063/1.468886

http://dx.doi.org/10.1063/1.468886

http://dx.doi.org/10.1021/jp971722o

http://dx.doi.org/10.1021/jp971722o

http://dx.doi.org/10.1038/355335a0



http://dx.doi.org/10.1016/0022-2313(93)90048-R

http://dx.doi.org/10.1021/j100081a025

http://dx.doi.org/10.1021/j100081a025

http://dx.doi.org/10.1073/pnas.93.13.6264


http://dx.doi.org/10.1038/nphoton.2009.187

http://dx.doi.org/10.1038/nphoton.2009.187


http://dx.doi.org/10.1002/bip.1974.360130102


[73] “Fluorescence correlation spectroscopy. II. an experimentalrealization”: D. L. Magde, E. L. Elson, W. W. Webb, Biopoly-mers 1974, 13, 29 – 61.

[74] “Fluorescence correlation spectroscopy. III. uniform trans-lation and laminar flow”: D. Magde, W. W. Webb, E. L. Elson,Biopolymers 1978, 17, 361 – 367.

[75] “Rotational brownian motion and fluorescence intensify fluctu-ations”: M. Ehrenberg, R. Rigler, Chem. Phys. 1974, 4, 390 – 401.

[76] “Fluorescence correlation spectroscopy as a probe of moleculardynamics”: S. R. Aragýn, R. Pecora, J. Chem. Phys. 1976, 64,1791 – 1803.

[77] R. Rigler, J. Widengren in Ultrasensistve Detection of SingleMolecules by Fluorescence Correlation Spectroscopy, Biosci-ence Third Conference (Eds.: B. Klinge, C. Owman), LundUniversity Press, Lund, Sweden, 1990, pp. 180 – 183.

[78] “Single-molecule fluorescence detection - auto-correlationcriterion and experimental realization with phycoerythrin”:K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl.Acad. Sci. USA 1989, 86, 4087 – 4091.

[79] “Detection of single fluorescent molecules”: E. Brooks Shera,N. K. Seitzinger, L. M. Davis, R. A. Keller, S. A. Soper, Chem.Phys. Lett. 1990, 174, 553 – 557.

[80] “Probing individual molecules with confocal fluorescencemicroscopy”: S. Nie, D. T. Chiu, R. N. Zare, Science 1994, 266,1018 – 1021.

[81] “Optical microscopic observation of single small molecules”: T.Hirschfeld, Appl. Opt. 1976, 15, 2965 – 2966.

[82] “Single molecules observed by near-field scanning opticalmicroscopy”: E. Betzig, R. J. Chichester, Science 1993, 262,1422 – 1425.

[83] “Single-molecule detection and photochemistry on a surfaceusing near-field optical-excitation”: W. P. Ambrose, P. M.Goodwin, J. C. Martin, R. A. Keller, Phys. Rev. Lett. 1994, 72,160 – 163.

[84] “Probing single-molecule dynamics”: X. S. Xie, R. C. Dunn,Science 1994, 265, 361 – 364.

[85] “Imaging and time-resolved spectroscopy of single molecules atan interface”: J. J. Macklin, J. K. Trautman, T. D. Harris, L. E.Brus, Science 1996, 272, 255 – 258.

[86] “Imaging of single fluorescent molecules and individual ATPturnovers by single myosin molecules in aqueous solution”: T.Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida,Nature 1995, 374, 555 – 559.

[87] “Imaging of single molecule diffusion”: T. Schmidt, G. J.Schutz, W. Baumgartner, H. J. Gruber, H. Schindler, Proc.Natl. Acad. Sci. USA 1996, 93, 2926 – 2929.

[88] Fluorescence Correlation Spectroscopy (Eds.: R. Rigler, E.Elson), Springer, Berlin, 2001.

[89] “Fluorescence correlation spectroscopy and its potential forintracellular applications”: P. Schwille, Cell Biochem. Biophys.2001, 34, 383 – 408.

[90] “Biological and chemical applications of fluorescence correla-tion spectroscopy: A review”: S. T. Hess, S. Huang, A. A.Heikal, W. W. Webb, Biochemistry 2002, 41, 697 – 705.

[91] “Fluorescence spectroscopy of single biomolecules”: S. Weiss,Science 1999, 283, 1676 – 1683.

[92] “Illuminating single molecules in condensed matter”: W. E.Moerner, M. Orrit, Science 1999, 283, 1670 – 1676.

[93] Single-molecule optical spectroscopy and imaging: From earlysteps to recent advances: W. E. Moerner, in Single MoleculeSpectroscopy in Chemistry, Physics and Biology: Nobel Sym-posium 138 Proceedings (Eds.: A. Graslund, R. Rigler, J.Widengren), Springer, Berlin, 2009, pp. 25 – 60.

[94] Single Molecule Spectroscopy: Nobel Conference Lectures(Eds.: R. Rigler, M. Orrit, T. Basche), Springer, Berlin, 2001.

[95] Single Molecule Spectroscopy in Chemistry, Physics and Biol-ogy: Nobel Symposium 138 Proceedings (Eds.: A. Gr�slund, R.Rigler, J. Widengren), Springer, Berlin, 2010.

[96] C. Zander, J. Enderlein, R. A. Keller, Single-Molecule Detec-tion in Solution : Methods and Applications, Wiley-VCH,Weinheim, 2002.

[97] C. Gell, D. J. Brockwell, A. Smith, Handbook of SingleMolecule Fluorescence Spectroscopy, Oxford Univ. Press,Oxford, 2006.

[98] Single-Molecule Techniques : A Laboratory Manual (Eds.: P. R.Selvin, T. Ha), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 2008.

[99] Handbook of Single-Molecule Biophysics (Eds.: P. Hinterdor-fer, A. M. van Oijen), Springer, New York, 2009.

[100] “Translational diffusion of individual class II MHC membraneproteins in cells”: M. Vrljic, S. Y. Nishimura, S. Brasselet, W. E.Moerner, H. M. McConnell, Biophys. J. 2002, 83, 2681 – 2692.

[101] “Cholesterol depletion suppresses the translational diffusion ofclass II major histocompatibility complex proteins in theplasma membrane”: M. Vrljic, S. Y. Nishimura, W. E. Moerner,H. M. McConnell, Biophys. J. 2005, 88, 334 – 347.

[102] “Cholesterol depletion induces solid-like regions in the plasmamembrane”: S. Nishimura, M. Vrljic, L. O. Klein, H. M.McConnell, W. E. Moerner, Biophys. J. 2006, 90, 927 – 938.PMCID: PMC1367117.

[103] “Single-molecule tracking”: M. Vrljic, S. Y. Nishimura, W. E.Moerner, Methods Mol. Biol. 2007, 398, 193 – 219.

[104] “Single-molecule nanoprobes explore defects in spin-growncrystals”: C. A. Werley, W. E. Moerner, J. Phys. Chem. B 2006,110, 18939 – 18944.

[105] “Simultaneous imaging of individual molecules aligned bothparallel and perpendicular to the optic axis”: R. M. Dickson,D. J. Norris, W. E. Moerner, Phys. Rev. Lett. 1998, 81, 5322 –5325.

[106] “Generating and exploiting polarity in bacteria”: L. Shapiro, H.McAdams, R. Losick, Science 2002, 298, 1942 – 1946.

[107] “Cell cycle regulation in Caulobacter : Location, location,location”: E. D. Goley, A. A. Iniesta, L. Shapiro, J. Cell Sci.2007, 120, 3501 – 3507.

[108] “Single molecules of the bacterial actin MreB undergo directedtreadmilling motion in caulobacter crescentus”: S. Y. Kim, Z.Gitai, A. Kinkhabwala, L. Shapiro, W. E. Moerner, Proc. Natl.Acad. Sci. USA 2006, 103, 10929 – 10934.

[109] “The bacterial actin MreB rotates, and rotation depends oncell-wall assembly”: S. van Teeffelen, S. Wang, L. Furchtgott,K. C. Huang, N. S. Wingreen, J. W. Shaevitz, Z. Gitai, Proc.Natl. Acad. Sci. USA 2011, 108, 15822 – 15827.

[110] “Contributions to the theory of the microscope and micro-scopic detection”: E. Abbe, Arch. Mikroskop. Anat. 1873, 9,413 – 468.

[111] “Breaking the diffraction resolution limit by stimulated emis-sion: Stimulated-emission-depletion fluorescence microscopy”:S. W. Hell, J. Wichmann, Opt. Lett. 1994, 19, 780 – 782.

[112] “Surpassing the lateral resolution limit by a factor of two usingstructured illumination microscopy”: M. G. L. Gustafsson, J.Microsc. 2000, 198, 82 – 87.

[113] “The role of molecular dipole orientation in single-moleculefluorescence microscopy and implications for super-resolutionimaging”: M. P. Backlund, M. D. Lew, A. S. Backer, S. J. Sahl,W. E. Moerner, ChemPhysChem 2014, 15, 587 – 599.

[114] “Imaging intracellular fluorescent proteins at nanometerresolution”: E. Betzig, G. H. Patterson, R. Sougrat, O. W.Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J.Lippincott-Schwartz, H. F. Hess, Science 2006, 313, 1642 – 1645.

[115] “Precise nanometer localization analysis for individual fluo-rescent probes”: R. E. Thompson, D. R. Larson, W. W. Webb,Biophys. J. 2002, 82, 2775 – 2783.


Chemie





http://dx.doi.org/10.1016/0301-0104(74)85005-6



http://dx.doi.org/10.1016/0009-2614(90)85485-U

http://dx.doi.org/10.1016/0009-2614(90)85485-U

http://dx.doi.org/10.1126/science.7973650


http://dx.doi.org/10.1364/AO.15.002965







http://dx.doi.org/10.1038/374555a0



http://dx.doi.org/10.1385/CBB:34:3:383

http://dx.doi.org/10.1385/CBB:34:3:383

http://dx.doi.org/10.1021/bi0118512



http://dx.doi.org/10.1016/S0006-3495(02)75277-6

http://dx.doi.org/10.1529/biophysj.104.045989


http://dx.doi.org/10.1021/jp057570b

http://dx.doi.org/10.1021/jp057570b




http://dx.doi.org/10.1242/jcs.005967

http://dx.doi.org/10.1242/jcs.005967

http://dx.doi.org/10.1073/pnas.0604503103




http://dx.doi.org/10.1007/BF02956173

http://dx.doi.org/10.1007/BF02956173

http://dx.doi.org/10.1364/OL.19.000780

http://dx.doi.org/10.1046/j.1365-2818.2000.00710.x

http://dx.doi.org/10.1046/j.1365-2818.2000.00710.x

http://dx.doi.org/10.1002/cphc.201300880


http://dx.doi.org/10.1016/S0006-3495(02)75618-X


[116] “Using photon statistics to boost microscopy resolution”: X.Michalet, S. Weiss, Proc. Natl. Acad. Sci. USA 2006, 103, 4797 –4798.

[117] “Optimized localization analysis for single-molecule trackingand super-resolution microscopy”: K. I. Mortensen, L. S.Churchman, J. A. Spudich, H. Flyvbjerg, Nat. Methods 2010,7, 377 – 381.

[118] “Diffusion of low density lipoprotein-receptor complex onhuman fibroblasts”: L. S. Barak, W. W. Webb, J. Cell Biol. 1982,95, 846 – 852.

[119] “Tracking kinesin-driven movements with nanometre-scaleprecision”: J. Gelles, B. J. Schnapp, M. P. Sheetz, Nature 1988,331, 450 – 453.

[120] “Real-time single-molecule imaging of the infection pathway ofan adeno-associated virus”: G. Seisenberger, M. U. Ried, T.Endress, H. Buning, M. Hallek, C. Braeuchle, Science 2001, 294,1929 – 1932.

[121] “Position measurement with a resolution and noise-limitedinstrument”: N. Bobroff, Rev. Sci. Instrum. 1986, 57, 1152 – 1157.

[122] W. Heisenberg, The Physical Principles of Quantum Theory,University of Chicago Press, Chicago, 1930, p. 22.

[123] “Myosin V walks hand-over-hand: Single fluorophore imagingwith 1.5-nm localization”: A. Yildiz, J. N. Forkey, S. A. McKin-ney, T. Ha, Y. E. Goldman, P. R. Selvin, Science 2003, 300,2061 – 2065.

[124] “The green fluorescent protein”: R. Y. Tsien, Annu. Rev.Biochem. 1998, 67, 509 – 544.

[125] “Three-dimensional imaging of single molecules solvated inpores of poly(acrylamide) gels”: R. M. Dickson, D. J. Norris,Y. L. Tzeng, W. E. Moerner, Science 1996, 274, 966 – 969.

[126] “Ultra-fast excited state dynamics in green fluorescent protein:Multiple states and proton transfer”: M. Chattoraj, B. A. King,G. U. Bublitz, S. G. Boxer, Proc. Natl. Acad. Sci. USA 1996, 93,8362 – 8367.

[127] “An optical marker based on the UV-induced green-to-redphotoconversion of a fluorescent protein”: R. Ando, H. Hama,M. Yamamoto-Hino, H. Mizuno, A. Miyawaki, Proc. Natl.Acad. Sci. USA 2002, 99, 12651 – 12656.

[128] “A photoactivatable GFP for selective photolabeling of pro-teins and cells”: G. H. Patterson, J. A. Lippincott-Schwartz,Science 2002, 297, 1873 – 1877.

[129] “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion”: J. Wiedenmann, S. Ivan-chenko, F. Oswald, F. Schmitt, C. Rçcker, A. Salih, K. Spindler,G. U. Nienhaus, Proc. Natl. Acad. Sci. USA 2004, 101, 15905 –15910.

[130] “Regulated fast nucleocytoplasmic shuttling observed byreversible protein highlighting”: R. Ando, H. Mizuno, A.Miyawaki, Science 2004, 306, 1370 – 1373.

[131] “Proposed method for molecular optical imaging”: E. Betzig,Opt. Lett. 1995, 20, 237 – 239.

[132] “3-dimensional super-resolution by spectrally selective imag-ing”: A. M. van Oijen, J. Kçhler, J. Schmidt, M. Mîller, G. J.Brakenhoff, Chem. Phys. Lett. 1998, 292, 183 – 187.

[133] a) “Far-field fluorescence microscopy beyond the diffractionlimit”: A. M. van Oijen, J. Kçhler, J. Schmidt, M. Mîller, G. J.Brakenhoff, J. Opt. Soc. Am. A 1999, 16, 909 – 915; b) “Ultra-high-resolution multicolor colocalization of single fluorescentprobes”: T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A.P. Alivisatos, S. Weiss, Proc. Natl. Acad. Sci. USA 2000, 97,9461 – 9466 .

[134] “High-resolution colocalization of single dye molecules byfluorescence lifetime imaging microscopy”: M. Heilemann,D. P. Herten, R. Heintzmann, C. Cremer, C. Mîller, P.Tinnefeld, K. D. Weston, J. Wolfrum, M. Sauer, Anal. Chem.2002, 74, 3511 – 3517.

[135] “Single-molecule high-resolution imaging with photobleach-ing”: M. P. Gordon, T. Ha, P. R. Selvin, Proc. Natl. Acad. Sci.USA 2004, 101, 6462 – 6465.

[136] “Nanometer-localized multiple single-molecule fluorescencemicroscopy”: X. Qu, D. Wu, L. Mets, N. F. Scherer, Proc. Natl.Acad. Sci. USA 2004, 101, 11298 – 11303.

[137] “Single molecule high-resolution colocalization of Cy3 and Cy5attached to macromolecules measures intramolecular distancesthrough time”: L. S. Churchman, Z. Oekten, R. S. Rock, J. F.Dawson, J. A. Spudich, Proc. Natl. Acad. Sci. USA 2005, 102,1419 – 1423.

[138] “Superresolution by localization of quantum dots using blink-ing statistics”: K. A. Lidke, B. Rieger, T. M. Jovin, R. Heintz-mann, Opt. Express 2005, 13, 7052 – 7062.

[139] “New directions in single-molecule imaging and analysis”: W. E.Moerner, Proc. Natl. Acad. Sci. USA 2007, 104, 12596 – 12602.

[140] “A non-gaussian distribution quantifies distances measuredwith fluorescence localization techniques”: L. Stirling Church-man, H. Flyvbjerg, J. A. Spudich, Biophys. J. 2006, 90, 668 – 671.

[141] “Sub-diffraction-limit imaging by stochastic optical reconstruc-tion microscopy (STORM)”: M. J. Rust, M. Bates, X. Zhuang,Nat. Methods 2006, 3, 793 – 796.

[142] “Ultra-high resolution imaging by fluorescence photoactivationlocalization microscopy”: S. T. Hess, T. P. K. Girirajan, M. D.Mason, Biophys. J. 2006, 91, 4258 – 4272.

[143] “Wide-field subdiffraction imaging by accumulated binding ofdiffusing probes”: A. Sharonov, R. M. Hochstrasser, Proc. Natl.Acad. Sci. USA 2006, 103, 18911 – 18916.

[144] “Controlled biomolecular collisions allow sub-diffraction lim-ited microscopy of lipid vesicles”: E. Mei, F. Gao, R. M.Hochstrasser, Phys. Chem. Chem. Phys. 2006, 8, 2077 – 2082.

[145] “Subdiffraction-resolution fluorescence imaging with conven-tional fluorescent probes”: M. Heilemann, S. van de Linde, M.Schîttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld,M. Sauer, Angew. Chem. Int. Ed. 2008, 47, 6172 – 6176; Angew.Chem. 2008, 120, 6266 – 6271.

[146] “Multicolor fluorescence nanoscopy in fixed and living cells byexciting conventional fluorophores with a single wavelength”:I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Mid-dendorf, J. Foelling, S. Jakobs, A. Schoenle, S. W. Hell, C.Eggeling, Biophys. J. 2010, 99, 2686 – 2694.

[147] “Resolving single-molecule assembled patterns with super-resolution blink-microscopy”: T. Cordes, M. Strackharn, S. W.Stahl, W. Summerer, C. Steinhauer, C. Forthmann, E. M.Puchner, J. Vogelsang, H. E. Gaub, P. Tinnefeld, Nano Lett.2010, 10, 645 – 651.

[148] “SPDM: Light microscopy with single-molecule resolution atthe nanoscale”: P. Lemmer, M. Gunkel, D. Baddeley, R.Kaufmann, A. Urich, Y. Weiland, J. Reymann, P. Mueller, M.Hausmann, C. Cremer, Appl. Phys. B 2008, 93, 1 – 12.

[149] “Super-resolution imaging in live caulobacter crescentus cellsusing photoswitchable EYFP: J. S. Biteen, M. A. Thompson,N. K. Tselentis, G. R. Bowman, L. Shapiro, W. E. Moerner, Nat.Methods 2008, 5, 947 – 949.

[150] “Superresolution imaging of targeted proteins in fixed andliving cells using photoactivatable organic fluorophores”: H. D.Lee, S. J. Lord, S. Iwanaga, K. Zhan, H. Xie, J. C. Williams, H.Wang, G. R. Bowman, E. D. Goley, L. Shapiro, R. J. Twieg, J.Rao, W. E. Moerner, J. Am. Chem. Soc. 2010, 132, 15099 –15101.

[151] “Enzymatic activation of nitro-aryl fluorogens in live bacterialcells for enzymatic turnover-activated localization microsco-py”: M. K. Lee, J. Williams, R. J. Twieg, J. Rao, W. E. Moerner,Chem. Sci. 2013, 4, 220 – 225.

[152] “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms”: F. Huang, T. M. P. Hartwich,F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil,


W. E. Moerner




http://dx.doi.org/10.1038/nmeth.1447


http://dx.doi.org/10.1083/jcb.95.3.846

http://dx.doi.org/10.1083/jcb.95.3.846

http://dx.doi.org/10.1038/331450a0

http://dx.doi.org/10.1038/331450a0



http://dx.doi.org/10.1063/1.1138619



http://dx.doi.org/10.1146/annurev.biochem.67.1.509

http://dx.doi.org/10.1146/annurev.biochem.67.1.509










http://dx.doi.org/10.1364/OL.20.000237

http://dx.doi.org/10.1016/S0009-2614(98)00673-3

http://dx.doi.org/10.1364/JOSAA.16.000909

http://dx.doi.org/10.1021/ac025576g

http://dx.doi.org/10.1021/ac025576g







http://dx.doi.org/10.1364/OPEX.13.007052



http://dx.doi.org/10.1038/nmeth929




http://dx.doi.org/10.1039/b601670g




http://dx.doi.org/10.1016/j.bpj.2010.08.012

http://dx.doi.org/10.1021/nl903730r

http://dx.doi.org/10.1021/nl903730r

http://dx.doi.org/10.1007/s00340-008-3152-x



http://dx.doi.org/10.1021/ja1044192

http://dx.doi.org/10.1021/ja1044192

http://dx.doi.org/10.1039/C2SC21074F


J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D.Toomre, J. Bewersdorf, Nat. Methods 2013, 10, 653 – 658.

[153] “Breaking the diffraction barrier: Super-resolution imaging ofcells”: B. Huang, H. Babcock, X. Zhuang, Cell 2010, 143, 1047 –1058.

[154] “Extending microscopic resolution with single-molecule imag-ing and active control”: M. A. Thompson, M. D. Lew, W. E.Moerner, Annu. Rev. Biophys. 2012, 41, 321 – 342.

[155] “Molecules and methods for super-resolution imaging”: M. A.Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, W. E. Moerner,Methods Enzymol. 2010, 475, 27 – 59.

[156] “Single-molecule and superresolution imaging in live bacteriacells”: J. S. Biteen, W. E. Moerner, Cold Spring HarborPerspect. Biol. 2010, 2, a000448.

[157] Springer Series on Fluorescence: M. D. Lew, S. F. Lee, M. A.Thompson, H. D. Lee, W. E. Moerner in Single-MoleculePhotocontrol and Nanoscopy, Springer, Berlin, Heidelberg,2012, pp. 1 – 24.

[158] “Microscopy beyond the diffraction limit using actively con-trolled single molecules”: W. E. Moerner, J. Microsc. 2012, 246,213 – 220.

[159] “Quantitative multicolor subdiffraction imaging of bacterialprotein ultrastructures in 3D”: A. Gahlmann, J. L. Ptacin, G.Grover, S. Quirin, A. R. S. von Diezmann, M. K. Lee, M. P.Backlund, L. Shapiro, R. Piestun, W. E. Moerner, Nano Lett.2013, 13, 987 – 993.

[160] “Super-resolution fluorescence imaging with single molecules”:S. J. Sahl, W. E. Moerner, Curr. Opin. Struct. Biol. 2013, 23,778 – 787.

[161] “Super-resolution microscopy approaches for live cell imag-ing”: A. Godin, B. Lounis, L. Cognet, Biophys. J. 2014, 107,1777 – 1784.

[162] “The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-terminal yellow fluorescent proteintag”: M. T. Swulius, G. J. Jensen, J. Bacteriol. 2012, 194, 6382 –6386.

[163] “A spindle-like apparatus guides bacterial chromosome segre-gation”: J. L. Ptacin, S. F. Lee, E. C. Garner, E. Toro, M. Eckart,L. R. Comolli, W. E. Moerner, L. Shapiro, Nat. Cell Biol. 2010,12, 791 – 798.

[164] “Super-resolution imaging of the nucleoid-associated proteinHU in caulobacter crescentus”: S. F. Lee, M. A. Thompson,M. A. Schwartz, L. Shapiro, W. E. Moerner, Biophys. J. 2011,100, L31 – L33.

[165] “Superresolution microscopy for microbiology”: C. Coltharp, J.Xiao, Cell. Microbiol. 2012, 14, 1808 – 1818.

[166] “Exploring bacterial cell biology with single-molecule trackingand super-resolution imaging”: A. Gahlmann, W. E. Moerner,Nat. Rev. Microbiol. 2014, 12, 9 – 22.

[167] “Single-molecule super-resolution imaging in bacteria”: D. I.Cattoni, J. B. Fiche, M. Noellmann, Curr. Opin. Microbiol.2012, 15, 758 – 763.

[168] “Fluorescent saxitoxins for live cell imaging of single voltage-gated sodium ion channels beyond the optical diffraction limit”:A. E. Ondrus, H. D. Lee, S. Iwanaga, W. H. Parsons, B. M.Andresen, W. E. Moerner, J. Du Bois, Chem. Biol. 2012, 19,902 – 912.

[169] “Sub-diffraction imaging of huntingtin protein aggregates byfluorescence blink-microscopy and atomic force microscopy”:W. C. Duim, B. Chen, J. Frydman, W. E. Moerner, ChemPhys-Chem 2011, 12, 2387 – 2390.

[170] W. C. Duim, “Single-molecule fluorescence and super-resolu-tion imaging of HuntingtonÏs disease protein aggregates”,Dissertation, Stanford, CA, Stanford University, 2012.

[171] “Cellular inclusion bodies of mutant huntingtin exon 1 obscuresmall fibrillar aggregate species”: S. J. Sahl, L. E. Weiss, W. C.Duim, J. Frydman, W. E. Moerner, Sci. Rep. 2012, 2, 1 – 7.

[172] “Small-molecule labeling of live cell surfaces for three-dimen-sional super-resolution microscopy”: M. K. Lee, P. Rai, J.Williams, R. J. Twieg, W. E. Moerner, J. Am. Chem. Soc. 2014,136, 14003 – 14006.

[173] “Three-dimensional super-resolution imaging by stochasticoptical reconstruction microscopy”: B. Huang, W. Wang, M.Bates, X. Zhuang, Science 2008, 319, 810 – 813.

[174] “High accuracy 3D quantum dot tracking with multifocal planemicroscopy for the study of fast intracellular dynamics in livecells”: S. Ram, P. Prabhat, J. Chao, E. S. Ward, R. J. Ober,Biophys. J. 2008, 95, 6025 – 6043.

[175] “Three-dimensional sub-100 nm resolution fluorescence mi-croscopy of thick samples”: M. F. Juette, T. J. Gould, M. D.Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T.Hess, J. Bewersdorf, Nat. Methods 2008, 5, 527 – 529.

[176] “Extending single-molecule microscopy using optical Fourierprocessing”: A. S. Backer, W. E. Moerner, J. Phys. Chem. B2014, 118, 8313 – 8329.

[177] “Three-dimensional, single-molecule fluorescence imagingbeyond the diffraction limit by using a double-helix pointspread function”: S. R. P. Pavani, M. A. Thompson, J. S. Biteen,S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, W. E. Moerner, Proc.Natl. Acad. Sci. USA 2009, 106, 2995 – 2999.

[178] “Three-dimensional localization precision of the double-helixpoint spread function versus astigmatism and biplane”: M.Badieirostami, M. D. Lew, M. A. Thompson, W. E. Moerner,Appl. Phys. Lett. 2010, 97, 161103.

[179] “Three-dimensional superresolution colocalization of intracel-lular protein superstructures and the cell surface in liveCaulobacter crescentus”: M. D. Lew, S. F. Lee, J. L. Ptacin,M. K. Lee, R. J. Twieg, L. Shapiro, W. E. Moerner, Proc. Natl.Acad. Sci. USA 2011, 108, E1102 – E1110.

[180] “Corkscrew point spread function for far-field three-dimen-sional nanoscale localization of pointlike objects”: M. D. Lew,S. F. Lee, M. Badieirostami, W. E. Moerner, Opt. Lett. 2011, 36,202 – 204.

[181] “A bisected pupil for studying single-molecule orientationaldynamics and its application to 3D super-resolution microsco-py”: A. S. Backer, M. P. Backlund, A. R. Diezmann, S. J. Sahl,W. E. Moerner, Appl. Phys. Lett. 2014, 104, 193701.

[182] “Optimal point spread function design for 3D imaging”: Y.Shechtman, S. J. Sahl, A. S. Backer, W. E. Moerner, Phys. Rev.Lett. 2014, 113, 133902.

[183] “Actin, spectrin, and associated proteins form a periodiccytoskeletal structure in axons”: K. Xu, G. Zhong, X.Zhuang, Science 2013, 339, 452 – 456.

[184] “Tracking single molecules at work in living cells”: A. Kusumi,T. A. Tsunoyama, K. M. Hirosawa, R. S. Kasai, T. K. Fujiwara,Nat. Chem. Biol. 2014, 10, 524.

[185] “A practical guide to single-molecule FRET”: R. Roy, S.Hohng, T. Ha, Nat. Methods 2008, 5, 507 – 516.

[186] “FRET in cell biology: Still shining in the age of super-resolution?”: H. E. Grecco, P. J. Verveer, ChemPhysChem2011, 12, 484 – 490.

[187] “Probing single biomolecules in solution using the anti-brownian electrokinetic (ABEL) trap”: Q. Wang, R. H. Gold-smith, Y. Jiang, S. D. Bockenhauer, W. E. Moerner, Acc. Chem.Res. 2012, 45, 1955 – 1964.

[188] “Single-molecule spectroscopy of photosynthetic proteins insolution: Exploration of structure – function relationships”:G. S. Schlau-Cohen, S. Bockenhauer, Q. Wang, W. E. Moerner,Chem. Sci. 2014, 5, 2933 – 2939.

[189] “Enzyme kinetics, past and present”: X. S. Xie, Science 2013,342, 1457 – 1459.

Received: March 2, 2015Published online: June 18, 2015


Chemie



http://dx.doi.org/10.1016/j.cell.2010.12.002

http://dx.doi.org/10.1016/j.cell.2010.12.002

http://dx.doi.org/10.1146/annurev-biophys-050511-102250

http://dx.doi.org/10.1101/cshperspect.a000448

http://dx.doi.org/10.1101/cshperspect.a000448

http://dx.doi.org/10.1111/j.1365-2818.2012.03600.x

http://dx.doi.org/10.1111/j.1365-2818.2012.03600.x

http://dx.doi.org/10.1021/nl304071h

http://dx.doi.org/10.1021/nl304071h

http://dx.doi.org/10.1016/j.sbi.2013.07.010

http://dx.doi.org/10.1016/j.sbi.2013.07.010



http://dx.doi.org/10.1128/JB.00505-12

http://dx.doi.org/10.1128/JB.00505-12

http://dx.doi.org/10.1038/ncb2083

http://dx.doi.org/10.1038/ncb2083



http://dx.doi.org/10.1111/cmi.12024

http://dx.doi.org/10.1016/j.mib.2012.10.007

http://dx.doi.org/10.1016/j.mib.2012.10.007

http://dx.doi.org/10.1016/j.chembiol.2012.05.021

http://dx.doi.org/10.1016/j.chembiol.2012.05.021



http://dx.doi.org/10.1021/ja508028h

http://dx.doi.org/10.1021/ja508028h




http://dx.doi.org/10.1021/jp501778z

http://dx.doi.org/10.1021/jp501778z



http://dx.doi.org/10.1063/1.3499652



http://dx.doi.org/10.1364/OL.36.000202

http://dx.doi.org/10.1364/OL.36.000202

http://dx.doi.org/10.1063/1.4876440


http://dx.doi.org/10.1038/nchembio.1558




http://dx.doi.org/10.1021/ar200304t

http://dx.doi.org/10.1021/ar200304t

http://dx.doi.org/10.1039/C4SC00582A




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