Reprinted from the November 2002 issue of Biophotonics International © Laurin Publishing Co. Inc.

A Laurin Publication

Choosing Filters for Fluorescence

Photonic Solutions for Biotechnology and MedicineNovember 2002

Confocal Microscopy Analyzes Cells

In living cells, protein activity often de-pends on their association in a com-plex of several molecules. For exam-

ple, the replication of chromosomes priorto cell division requires the cooperationof proteins such as DNA polymerases, he-licases and ligases to build the replicationmachinery. Furthermore, most enzymesneed to interact with co-factors to becomeactive. For instance, protein kinase C is acrucial enzyme that influences a greatnumber of cellular processes, includingsecretion, cell growth and differentiation.The interaction of the kinase with specificlipid co-factors is essential for the regu-lation of its activity.

To study when and where proteins as-sociate with each other, scientists com-monly label them with different fluoro-phores and subject them to fluorescencemicroscopy. Labeling can be either indi-rect, by using fluorescently labeled anti-bodies that are directed against the pro-tein of interest, or direct via conjugationbetween protein and fluorophore. A pop-ular method for the latter approach is the

SUMMARY

When and where specific pro-teins interact with each otheris the central question thatneeds to be resolved to gain abetter understanding of theirfunction. Confocal laser scan-ning microscopes can supportkey approaches to the study offunctional interactions of bio-molecules.

by Dr. Richard Ankerhold and Dr. Bernhard Zimmermann

Confocal MicroscopyReveals Molecular Interactions

CELL ANALYSIS

Figure 1. Individual pinholes in front of every detector (P1-P4), including the Metadetector, allow the user control of confocal slice thickness for each channel.

genetic engineering of fu-sion proteins composedof the protein of interestand green fluorescent pro-tein (GFP) or its variants.The fusion construct canthen be expressed in cells,tissues or even whole an-imals.

A major advantage ofconfocal methods overconventional fluorescencemicroscopy is the abilityto optically section cellsand tissue in three di-mensions without me-chanically disrupting thestructure of interest. In ad-dition, a laser scanningmicroscope allows theuser to fully control thedepth of focus and elim-inate unwanted light fromout-of-focus areas.Quantitative collocaliza-tion assays, fluorescenceresonance energy transfer(FRET) microscopy andfluorescence correlationspectroscopy are all tech-niques that can be usedwhere confocal methodsare beneficial or essentialto investigate the func-tional interaction of mol-ecules.

Identifying collocalizationIn collocalization experiments, the co-

incidence of two fluorescently labeledproteins in the same spot is indicative oftheir potential interaction. Hereby, con-focal technology virtually excludes thepossibility of false-positive observationsthat can result from the low depth of focusof conventional fluorescence microscopy.

Collocalization studies require identi-cal dimensions for the detection volumesfrom which the two signals are collected.Most importantly, the confocal sectionsmust exhibit identical thicknesses; other-wise, the reconstructed 3-D image willshow overlapping parts that do not over-lap in reality. Also, the two fluorescentsignals indicating the position of the pro-teins must be completely and unam-biguously separated, each in its own imag-

ing channel. Finally, the method requiresadvanced software functions specializedfor quantitative analysis of the collocal-ization data.

This may sound simple, but it is not.The thickness of confocal image slices de-pends upon the excitation and emissionof the dyes used, as well as on the size ofthe confocal pinhole; the pinhole canhelp compensate for differences in slicethickness that result from different-col-ored dyes. However, this requires a mul-tipinhole system, so that the user can ad-just the optical slice thickness for eachcolor/detector individually. The LSM 510series from Carl Zeiss provides the re-quired architecture with individual mo-torized pinholes in front of every detectorand automatic adjustment routines(Figure 1). The software allows the re-searcher to adjust the pinholes of eachconfocal channel to the same optical

thickness, independent ofthe color.

Complete separation ofthe fluorescent signals re-quires careful selection ofthe fluorescent dyes for acollocalization experiment.Because of the spectralproperties of dyes, the emis-sion of one dye typicallyoverlaps the emission of thesecond. This effect is pres-ent in most combinationsof fluorescent dyes, even forthe classical combinationof FITC and rhodamine.Simultaneous excitationand detection of these dyescauses the green fluores-cence emission to con-tribute to the detection ofthe red, a phenomenonknown as crosstalk. One so-lution implemented in themicroscope, multitracking,allows the user to alternatebetween excitation lines inan extremely fast mannerthat can be applied to live-cell measurements. Thisprocedure can minimizecrosstalk and may eveneliminate it if the excitationspectra of the dyes do notoverlap.

However, this doesn’t eliminate crosstalkfor all experiments because some studies re-quire fluorophores with overlapping emis-sion and excitation spectra, such as com-binations of GFP and its variants.

To address this issue, emission finger-printing, a detection technology incor-porated into the LSM 510 Meta, can sep-arate the spectra even if the overlap is ex-treme. The system allows the user tocompletely separate fluorescence emis-sions even if much of the emission pro-files overlap, such as green and yellowfluorescent protein or GFP and SytoxGreen.

Emission fingerprinting requires theacquisition of the spectral composition ofthe fluorophores’ emission signatures inthe sample and mathematical separationinto images representing the distributionof the individual fluorophores (Figure3). An algorithm allows the user to au-

Figure 2. A confocal image of a 6-day-old zebra fish (dorsal view)embryo that has been double-labeled with antibodies against celladhesion molecules shows staining of different subpopulations of axonsin the nervous system. Yellow indicates areas of collocalization, whileother axons show either green or red fluorescence. Courtesy of M.Marx, University of Konstanz, Germany, and M. Bastmeyer, Universityof Jena, Germany.

tomatically extract the spec-tral components. A highly par-allel acquisition schememakes emission fingerprint-ing ideal for live-cell imaging,particularly in the onlinemode where the separationoccurs instantaneously dur-ing data acquisition.

“We are anxious to move intomulticolors,” said William C.Hyun, director of theLaboratory of Cell Analysis atthe University of California, SanFrancisco. “The ability to tuneemissions and pick out the spe-cific pattern of these fluorescentproteins allows us to go in andtease apart multiple probe setsfor multiple GFPs. Being ableto clear out the com-plicated spectral over-laps is a tremendousbenefit.“

Key to successAccurate three-di-

mensional detectionwith a multiple pin-hole system and un-ambiguous separationof spectrally overlap-ping dyes are the firstprerequisites for a suc-cessful collocalizationexperiment. Such stud-ies also require soft-ware functions for

analysis of the resultingimage. The latest versionof the LSM 5 software(Rel. 3.2) introduces acollocalization analysispackage (Figure 4).

Key is the interactivelink of the image displaywith a two-dimensionalscatter plot representingthe distribution of indi-vidual voxels of theimage according to thesignal intensities of theimage channels. Eitherin the image display orin the scatter plot, re-gions can be selected in-teractively for analysis.A comprehensive set ofquantitative data, in-cluding various collo-calization coefficientsand correlation param-eters, can be directly ex-tracted from the selectedregions. With this, theuser can adjust collocal-ization thresholds ac-cording to backgroundintensities within theimages or can perform,visualize and quantita-tively assay for collocal-ization in the image setand color-overlay imagevoxels that are positivelytested for collocaliza-tion.

The optical resolutionof light microscopes, includingconfocal microscopes, limits thedetection of protein proximitiesto about 0.2 µm in conventionalcollocalization experiments.However, studying the physicalinteractions of protein partnersrequires higher resolution.

FRET can resolve the proxim-ity of proteins beyond the opti-cal limit of light microscopes (in

Figure 3. For emissionfingerprinting of GFP andyellow fluorescent protein incultured cells, the useracquires the spectralemission signature in a seriesof spectrally resolved images(lambda stack) followed bylinear unmixing on the basisof reference spectra for theproteins. The result is a pairof images representing theGFP and yellow fluorescentprotein distribution in thesample, respectively. Samplecourtesy of Frank D. Böhmer,Friedrich Schiller University.

Figure 4. The software allowsthe user to performquantitative collocalizationanalysis.

CELL ANALYSIS

the range of 1 to 10 nm). FRET imagingmeasures the nonradiative transfer of pho-ton energy from an excited fluorophore(the donor) to another fluorophore (theacceptor). This interaction results inquenching of donor fluorescence and si-multaneous sensitized photon emissionby the acceptor molecule. Its efficiency de-pends on various parameters, including asufficient overlap of donor emission andacceptor excitation spectra (as in cyan andyellow fluorescent proteins, today the mostwidely used FRET pair).

The amount of energy transferred de-pends on the distance between the donorand acceptor. Therefore, the techniquecan be used as a molecular ruler and as anindicator of physical interactions betweenfluorescently labeled molecules. The useof pairs of fluorescent proteins, such ascyan fluorescent protein and yellow fluo-rescent protein as donor and acceptormolecules, respectively, has greatly facil-itated in vivo FRET studies because theyallow labeling of the molecules of inter-est with excellent specificity while re-taining the integrity of the cell and itsphysiological functions.

Several methods have been developedfor imaging FRET in cells. One approachinvolves either bleaching the acceptor mol-ecule and subsequent monitoring of con-comitant dequenching of donor fluores-cence or examining relative temporalchanges of donor emission and accep-tor/FRET signals under donor excitationin dynamic situations (Figure 5). An al-

Figure 5. Emission fingerprinting reveals complete bleaching of yellow fluorescent protein and concomitant dequenching ofcyan fluorescent protein, indicating fluorescence resonance energy transfer in the bleached areas (marked by dashes).

Figure 6. By combining a confocal laser scanning microscope and a fluorescencecorrelation spectrometer, structures can be made visible and, at the same time,molecular interactions can be investigated.

CELL ANALYSIS

ternative approach uses various excitation-emission configurations (such as donorexcitation-donor emission, acceptor exci-tation-acceptor emission, donor excita-tion-acceptor emission) and subsequentevaluation of sensitized acceptor emissionvia mathematical correction. The LSM 510’sabilities, such as multitracking, pixel-pre-cise bleaching and online ratiometricanalysis, allow the user to perform the de-scribed types of FRET experiments withhigh three-dimensional resolution.

Until now, however, acceptor and donorwith overlapping excitation and emissionspectra required that the user select nar-row detection bands to differentiate be-tween emissions. Even then, quantitativeapproaches had to include complex math-ematical corrections to account for in-evitable crosstalk between channels andfor local concentration differences.

Emission fingerprinting overcomes sev-eral of these drawbacks. Parallel acquisitionof the emissions from donor and acceptorprovide the high photon efficiency andthe image acquisition speed required inlive-cell FRET microscopy. Sorting donor

and acceptor emissions into separate out-put channels can produce significantlybigger signal changes in dynamic FRET sit-uations. Finally, this method eliminatesthe need to correct for emission crosstalkin quantitative studies.

While collocalization and FRET exper-iments still measure the distance betweenthe protein partners of interest as an in-dicator of their possible interaction, fluo-rescence correlation spectroscopy directlymeasures the associated changes of dif-fusion times. We have combined our sys-tem with this technique in one instru-ment dedicated to fluorescence correla-tion spectroscopy analysis in living cells.

Single-molecule sensitivityFirst, the researcher acquires a 3-D

image stack of the cell. Then several fluo-rescence correlation spectroscopy analy-sis spots can be placed anywhere in oroutside the cell at X, Y and Z positions.Each spot represents a measurement vol-ume of less than 10215 l. When startingthe measurement, the system excites thespot and measures the fluorescently la-

CELL ANALYSIS

beled biomolecules passing through thedefined volume as a photon shower of adefined time (Figure 6). From this, thesystem can calculate the characteristic dif-fusion times of the proteins of interest. Ifthe instrument detects a prolonged dif-fusion time, the molecule of interest isdirectly interacting with another.

A software correlator analyzes the pho-tons registered by the detector for timeinterrelations. Thereby, the instrumentcan distinguish between populations ofbiomolecules with different diffusiontimes; e.g., populations of single mole-cules and a population of molecules as-sociated in a complex. High-sensitivityavalanche photodiodes allow it to detectsingle molecules, providing a tool for di-rect measurement of dynamic molecularinteractions within living cells. G

Meet the authorsRichard Ankerhold is head of the Training,

Application and Support Center, and BernhardZimmermann is product manager for confocallaser scanning and advanced imaging micros-copy at Carl Zeiss GmbH in Jena, Germany.