B I O G R A P H YSimon Schlachter recei-ved his BS (2004) and MS(2006) in electrical engi-neering from the Univer-sity of Illinois at Urbana-Champaign, USA. He iscurrently pursuing hisPhD at the University of Cambridge in theLaser Analytics Group working on the devel-opment of advanced optical microscopytechniques (http://laser.cheng.cam.ac.uk).

A B S T R A C TThe advent of novel supercontinuum lightsources is set to change the future of confo-cal microscopy, enabling wavelength- andlifetime-resolved imaging. Here we describethe development of a versatile confocalmicroscopy system which features full flexi-bility for wavelength excitation and spec-trally resolved fluorescence detection. Thesystem makes use of a compact, turn-keysupercontinuum light source and a custombuilt prism spectrometer in combinationwith a commercial microscope frame. Itgreatly expands the parameter space acces-sible to the microscopist and permits the res-olution and differentiation of features thatare spectrally similar. Some of these capabil-ities are demonstrated with applications inplant and live-cell samples.

K E Y W O R D Slight microscopy, confocal microscopy, fluo-rescence microscopy, supercontinuum radia-tion, laser, spectral imaging, live-cell

A C K N O W L E D G E M E N T SWe acknowledge the support of OlympusUK in this research. Funding for this researchwas obtained from the UK’s Engineeringand Physical Sciences Research Council,under grant GR/R98679/01. CFK acknowl-edges funding from the Leverhulme Trustand the SAOT school in advanced opticaltechnologies, Erlangen, Germany.

A U T H O R D E TA I L SDr Clemens Kaminski, University of Cambridge, Department ofChemical Engineering, Pembroke Street,Cambridge CB2 3RA, UKTel: +44 (0)1223 763135Email: cfk23@cam.ac.uk

Microscopy and Analysis 22(3):11-13 (EU),2008

CO N F O C A L MI C R O S C O P Y

I N T R O D U C T I O NResearchers are continually seeking toimprove methods for the quantification of afluorophore’s local environment and func-tional state. This is being achieved by usinghyperspectral, multi-modal imaging tech-niques. For example, combined measurementsof fluorescence excitation and emission spec-tra as well as lifetimes provide a new capabil-ity for determining the photophysical state ofthe fluorophore. This state is affected by thelocal environmental properties of the fluo-rophore, such as pH, viscosity, and the pres-ence of quenchers. Thus, by precisely measur-ing the photophysical state of a fluorophore,we inherently learn about the environment ofthat fluorophore. In biology this has led to arevolution: much of what we know aboutchemical pathways in living cells stems fromdata generated by sophisticated fluorescencemicroscopy techniques.

The advent of supercontinuum sources inthe late 1990s provided for the first time abroadband excitation source having a contin-uous spectral range from the near-UV to thenear-infrared with power densities that are onthe order of 1 mW nm-1. The use of acousto-optic tunable filters (AOTFs) in conjunctionwith this wide spectrum creates an idealsource for fluorescence microscopy excitation,capable of rapidly and continuously scanningthrough a wide range of wavelengths.

Access to a continuous spectrum is especiallyuseful for several new classes of fluorophore.Of particular interest are the recently devel-oped fluorescent monomeric proteins, such asthe mFruit fluorophores. These fluorescentlabels are small enough to avoid interferingwith the protein(s) under investigation, yethave relatively high quantum efficiency, good

photostability and a wide range of emissionwavelengths [1]. The peak excitation wave-lengths of these new fluorescent proteins,however, often lie between the fixed laserlines that are available with traditional fluo-rescence microscopes. For example, mCherryhas a peak excitation wavelength of 587 nm,which is between the 543 nm and the 633 nmHe:Ne lines. The supercontinuum laser cou-pled with an AOTF enables efficient excitationof the mCherry fluorophore at the peakabsorption wavelength.

With such a flexible excitation source, it isimportant to have an equally capable detec-tion system that can provide spectrallyresolved measurements of the fluorescenceemission over a wide bandwidth. Combiningspectrally resolved excitation and detectionsystems opens new frontiers of imaging possi-bilities. For example, several fluorophores canbe individually excited and detected whilstminimizing cross-excitation and separatelyresolving each fluorophore. The use of multi-ple fluorophores is becoming increasinglyimportant in the study of the complex interac-tions of proteins or intracellular structures [2,3].

Additionally, spectrally resolved microscopyis useful for studying the Förster resonanceenergy transfer (FRET) phenomenon. Subtlespectral emission changes caused by FRETbetween two fluorophores can be fully quan-tified by the detector, and undesirable excita-tion of the acceptor fluorophore minimized bythe highly specific supercontinuum-AOTFsource [2].

We expect that spectrally resolvedmicroscopy will become an indispensable toolas researchers seek to extract ever more infor-mation from fluorescence measurements.

In this article we describe the integration of

Spectrally Resolved Confocal FluorescenceMicroscopy with a Supercontinuum Laser S. Schlachter,1 A. Elder,1 J. H. Frank,2 A. Grudinin,3 C. F. Kaminski1

1. Dept. of Chemical Engineering, University of Cambridge, UK. 2. Combustion Research Facility, Sandia National Laboratories, Livermore, CA, USA. 3. Fianium Laser Systems, Southampton, UK

Figure 1: (a) Visible spectrum from the Fianium SC450 supercontinuum laser with different pumping intensities. (b) Power transmitted by an acousto-optical tunable filter for a single line with full incident laser power.

MICROSCOPY AND ANALYSIS MAY 2008 11

a b

a supercontinuum laser into a confocal imag-ing microscope that was developed in theDepartment of Chemical Engineering at theUniversity of Cambridge. The supercontinuumsource is generated by a compact fibre laser-pumped photonic crystal fibre.

S U P E R C O N T I N U U M S O U R C EThe word ‘supercontinuum’ refers to thebroad optical spectrum that is generated bypumping a nonlinear optical fibre with a short,high-intensity laser pulse. Supercontinuumgeneration involves a combination of nonlin-ear optical effects, including soliton genera-tion, self-phase modulation (SPM), wave mix-ing, and Raman scattering. Their relativeimportance depends on the type of pumpsource.

Photonic crystal fibres are frequently used inthe generation of supercontinua because oftheir highly nonlinear properties and the rela-tive ease of coupling light into them [4]. Tradi-tionally, high-peak power solid state pulsedlasers such as Nd:YAG and Ti:Sapphire havebeen used to pump these fibres. Recently,amplified mode-locked fibre lasers with shortpulse widths and high peak powers havebecome available for supercontinuum genera-tion [5]. The direct coupling of these fibrelasers into photonic crystal fibres provides anentirely fibre-based supercontinuum sourcethat is robust, reliable, and can be packaged ina turn-key commercial system.

Our supercontinuum source (Fianium SC450,Fianium Inc.), uses a mode-locked fibre laser (l= 1060 nm) as a master oscillator. The pulsesproduced by the master oscillator are opticallyamplified using a Yb-doped double-clad,pumped fibre. The amplified pulses propagatethrough a photonic crystal fibre in which theaforementioned nonlinear optical effectscause significant broadening of the pulse spectrum.

The visible portion of the supercontinuumspectrum is shown in Figure 1. The nearinfrared region of the spectrum is removedbut is available for use with a different AOTFcrystal.

Advances in supercontinuum sources con-tinue to expand their output to shorter wave-

lengths, and currently wavelengths down to400 nm are accessible [6]. This allows the vastmajority of currently available fluorophores tobe excited with a single source.

S Y S T E M D E S I G NOur group has built a microscope system forspectrally resolved confocal fluorescenceimaging by coupling a supercontinuum laserand spectrometer to a commercial OlympusIX70 frame with an FV300 confocal scanninghead. A block diagram of the microscopesetup is shown in Figure 2.

The supercontinuum source described in theprevious section is coupled to an AOTF thatenables electronic selection of a 1nm-wideband of the supercontinuum spectrum. TheAOTF consists of a TeO2 anisotropic crystal thatis driven by an ultrasonic transducer. Theacoustic waves propagating in the crystal cre-ate a periodic modulation of the refractiveindex. This periodic modulation acts as a Bragggrating, causing the deflection of a narrowband of wavelengths that match the periodicspacing of the grating. The wavelength andintensity of the deflected beam are controlledby varying the transducer driving frequencyand amplitude, respectively.

The output beam from the AOTF is coupledinto a commercial Olympus FV300 confocalscan head, which directs the beam into themicroscope objective lens via a pair of scan-ning mirrors. This confocal scanner is con-trolled through proprietary Olympus software

that provides a wide range of options for sam-ple illumination, including region-of-interestscanning, zoom-in, and Z stacks. Fluorescenceemission from the sample is imaged onto theconfocal pinhole and is subsequently coupledinto a custom-designed, high-throughputspectrometer.

The prism spectrometer spatially dispersesthe fluorescence spectrum onto a photomulti-plier tube (PMT). Two independently-con-trolled knife-edges placed in front of the PMTcontrol which region of the spectrum reachesthe PMT. The prism and lenses in the spec-trometer are anti-reflection coated for the vis-ible spectrum, resulting in 80% transmissionof the spectrometer system. Both the spec-trometer slit position and the AOTF drivingfrequency and amplitude are controlled via asingle LabView interface, which is synchro-nized with the confocal scanner.

This allows us to quickly set up excitationscans, in which the excitation wavelength israpidly swept across a given range while thesample is continually imaged. Emission scansare also possible, and the two can be com-bined in a joint excitation-emission scan wherethe fluorescence spectrum is sampled for eachexcitation wavelength at each pixel in theimage, providing a large amount of hyper-spectral data.

The entire system has been carefully cali-brated to correct for the effects of variations inthe excitation laser power, detection band-width, and detector sensitivity [7].

Figure 2: Block diagram of a microscope system for spectrally resolved confocal fluorescence imaging.

MICROSCOPY AND ANALYSIS MAY 200812

Figure 3: Two images of Convallaria majalis taken at excitation wavelengths of (a) 620 nm and (b) 530 nm. Each image is 355 µm x 355 µm. (c) A plot of fluorescence intensity versus excitation wavelength for several different structures within the sample (see (a) for locations).

CO N F O C A L MI C R O S C O P Y

R E S U LT STo demonstrate the capabilities of this spec-trally resolved confocal microscope, weimaged a fixed and stained section of Conval-laria majalis. The sample was stained withsafranin and fast-green dyes, which have exci-tation peaks at 530 nm and 620 nm, respec-tively.

We performed an excitation scan, rangingfrom 480 nm to 640 nm whilst continuallyacquiring 2D confocal images. Two imagesfrom this sequence, taken at 530 nm and 620nm are shown in Figure 3. Different structuresare evident at the two excitation wavelengths,indicating that the different stains bind pref-erentially to different structures within thesample.

A full excitation spectrum is available ateach pixel in the image, and several of thesespectra are shown in Figure 3c. As can be seen,the ability to resolve the entire excitationspectrum allows a very precise pixel-by-pixeldifferentiation of the multiple stains withinthe sample.

Though the microscope has been exten-sively modified, it still retains its original point-spread function, and thus the optical resolu-tion of the instrument is unchanged. This isdemonstrated in the high-resolution 2D and3D fluorescence measurements in Figure 4.The imaged volumes in Figures 4 c-e are 92 µm3 138 µm 3 18 µm with a voxel size of 0.46 30.46 3 0.70 µm3. Features of the sample arestill clearly resolved at this magnification, andthe PSF of the microscope was measured to be330 nm (lateral), 680 nm (axial) for a 490 nmillumination wavelength, in line with othercommercial instruments.

To demonstrate that our instrument is fastenough to image live cells, we performed afluorescence excitation scan of human U2OSosteosarcoma cells that had been transfectedwith a plasmid expressing an mCherry-Rad51fusion protein. Rad51 is a DNA binding pro-tein that is involved in DNA repair, andmCherry is a fluorescent monomeric proteindescribed earlier. The excitation wavelengthwas varied between 537 nm to 607 nm in four-teen steps, with a 5123512 pixel imageacquired for each step. The results of this exci-tation scan are shown in Figure 5. As expected,the excitation spectrum peaks at 587 nm,which is not accessible with gas or diode lasers.The closest commonly available laser line is the543 nm He:Ne line, which from Figure 5dwould result in a 60% decrease in fluores-cence. Due to the overexpression of Rad51,long fibrils of DNA wrapped in fluorescentRad51 protein appear in the nucleus of thecell. These fibrils are below the resolvable limitof the instrument and thus have been blurredby the PSF, which gives a good idea of the res-olution of the instrument.

F U T U R E D E V E L O P M E N T SWe will shortly be adding the capability forspectrally resolved fluorescence lifetime mea-surements to our confocal microscope. Thesupercontinuum source is ideally suited forlifetime imaging, because it produces 5 pspulses at a 40 MHz repetition rate. Further-

more, the AOTF allows the excitation power tobe quickly and accurately varied, and thus withthe addition of a time-correlated single-pho-ton counting (TCSPC) card to the PMT output,it is straightforward to integrate fluorescencelifetime imaging into the setup. The wave-length selectivity of the detection system andthe excitation source will be preserved, so thatthe lifetime of the fluorophore can be deter-mined at different spectral ranges.

As an additional imaging modality, we arealso adding a second detection channel, polar-ized orthogonal to the first, to allowanisotropy imaging. Our instrument will thenbe capable of eight dimensional imaging (x, y,z, t, lifetime, lambda excitation, lambda emis-sion and anisotropy). With these additions wehope to create a truly multi-modal confocalmicroscope, capable of quantifying all aspectsof fluorescence emission.

R E F E R E N C E S1. Shaner, N. C. et al. Improved monomeric red, orange and

yellow fluorescent proteins derived from Discosoma sp. redfluorescent protein. Nat. Biotech. 22(12):1567-1572, 2004.

2. Zimmermann, T. et al. Spectral imaging and its applicationsin live cell microscopy. FEBS Lett. 546:87-92, 2003.

3. Stephens, D. J. et al. COPI-coated ER-to-Golgi transportcomplexes segregate from COPII in close proximity to ERexit sites. J.Cell Sci. 113:2177-2185, 2000.

4. Russell, P. Photonic Crystal Fibres. Science 299:358-362, 2003.5. Okhotnikov, O. G. et al. Mode-locked ytterbium fiber laser

tunable in the 980–1070-nm spectral range. Optics Letters28(17):1522-1524, 2003.

6. Clowes, J. Supercontinuum sources head for medicalmarket. Optics and Laser Europe 153:19-22, 2007.

7. Frank, J. H. et al. White Light Confocal Microscope forSpectrally Resolved Multi-dimensional Imaging. J.Microscopy 227(3):203-215, 2007.

©2008 John Wiley & Sons, Ltd

Figure 4:High-resolution confocal (a) fluorescence and (b) broadband transmission images of Convallaria rhizome. The colour scale for (a) is the same as in Fig-ures 3 a,b. (c-e) Comparison of 3D confocal fluorescence measurements of Convallaria with excitation wavelengths at 500, 540, and 580 nm. Theimaged volume is 92 µm x 138 µm x 18 µm.

MICROSCOPY AND ANALYSIS MAY 2008 13

Figure 5:(a-c) Images of a human U2OS osteosarcoma cell labelled with mCherry-Rad51 fluo-rescent protein. Excitation wavelength was varied from 537 nm to 607 nm, and eachimage dimension is 59 µm x 59 µm. Excitation wavelengths are indicated in eachimage. (d) mCherry-Rad51 fluorescence excitation spectrum from a region within the cell.