Imaging of Biological Systems with Fluorescent Technologies

16 SCIENCE & MEDICINE

Imaging of Biological Systems withFluorescent Technologies

Fluorescence microscopyimaging has become one ofthe most useful techniques

in biomedical research, usedto assess the activity of

individual cells as well assubcellular processes. Thepast 2 decades have seen

tremendous advances in therational design and develop-

ment of probes to serve asreporters of the intracellular

environment of cells. Newfluorescent molecules have

been engineered to followsignaling traffic, sense ionic

and nonionic secondary messengers, and report

various kinase activities. In particular, the advent of

GFP cloning has provided ameans of making specific

proteins visible in living cells,allowing the study of

molecular behavior and interactions with high spatialand temporal resolution. It is

now possible to examinethese events in intact tissue,

enabling a more completeunderstanding of complex

physiologic processes.SCI & MED 10(1):16-29, 2005.

T he predominant focus ofbiomedical research in thepost-genomic era is direct-

ed toward identifying the functionof molecules at the cellular andsubcellular levels in living systems,such that an integrated under-standing of their function is possi-ble. This goal demands both tem-poral and spatial analysis that issimply not possible using methodsdependent on the extraction or dis-ruption of cells in which the eventsof interest are occurring.

Fortunately, the “post-genomic”need to study molecular functionhas been paralleled by extraordi-nary progress in optical microscopyand computer technologies. Con-temporary live cell microscopy canbe used to examine the expression,functional role(s), and interactionsof multiple molecules concurrentlywithin the cellular environment.Also, new methods allow the exam-ination of these events in intact tis-sue in real time, enabling a greaterunderstanding of cellular interac-tions and their interdependence.

The field of fluorescence micro-scopy has seen the principal ad-vances. The development of newmicroscopic methods, coupled withthe use of fluorescent proteins, newfluorescent dye technologies, high-ly sensitive detectors, and inexpen-sive powerful computers, have madethis technique widely used in bio-medical research. This review out-lines the principles, potential ap-plication, and implementation ofcontemporary fluorescent imagingtechniques in living cell systems.

Electron Excitation andRelaxation Produce

Fluorescence

Fluorescence microscopy dependson the inherent ability of a fluores-cent molecule (fluorophore) to beexcited by, and to emit light of, aspecific and known wavelength.Generally, the excitation wave-length is shorter (more blue) thanthe emission wavelength.

If light is considered as photonicpackets rather than electromag-netic waves, the process of fluores-cence excitation can be considereda quantum event. Incident photonsof a specific wavelength excite elec-trons within the fluorophore, gen-erally in double bonds or pi orbit-als, from the ground state to a de-fined higher-energy singlet state.The energy- or wavelength-selec-tive nature of this transition occursbecause electrons can only movebetween defined energy states withspecific electrical, vibrational, androtational components.

The time of electron excitation isshort, in the femtosecond range.However, while the electron is inthis excited state, it loses energyvia internal conversion. Subse-quently, the electron relaxes backto the ground state and releases alower-energy or longer wavelengthphoton.

The difference in wavelength be-tween the excitation and emissionlight is known as the Stokes shiftand is the essential principle in allfluorescence measurements. TheStokes shift allows emission pho-

Claudette M. St. Croix, Bruce R. Pitt, and Simon C. Watkins

Publication date: 7 July 2005

tons to be differentiated from exci-tation photons and the backgroundof nonfluorescing molecules.

It also should be mentioned thatdipole changes affecting the ioniccharge balance within the moleculemay occur, causing a shift in exci-tation or emission wavelengths.Generally, such changes are due tochanges in the solvent characteris-tics (more or less polar), includingincreases in specific ionic species(e.g., Ca2+). These wavelengthchanges may be measured and arethe principle behind ratiometricdyes, such as the calcium indicatorFura-2, which changes excitationmaxima on binding free calcium.With appropriate controls (chelat-ing all calcium or flooding the sys-tem with calcium in the case ofFura-2), it is possible to makehighly accurate measurements ofionic concentrations.

Instrumentation for Live Cell Imaging

Controls Phototoxicity

Traditional microscopic tools, aswith biochemical and/or molecularbiology approaches, provide infor-mation as isolated static snapshots.To generate temporally useful datawith these systems, it is necessaryto perform multiple experiments at

HIGH-ENERGYSINGLET STATE LOW-ENERGY

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GREG GAMBINO

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Fluorescence results from a three-stage process. A photon ofenergy hυEX is supplied by an external source (e.g., ultravioletlamp or laser) and is absorbed by the fluorophore, exciting anelectron to jump to a higher energy level (singlet state S1'). Theexcited state exists for a short time (typically 1–10 nsec) andthe energy of S1' is partially dissipated (to S1). A lower-energyphoton hυEM is emitted, producing the fluorescence, as the flu-orophore returns to its ground state S0.

Stokes shift refers to the loss of vibrational energy when elec-trons relax from the excited state back to the ground state. Asa result of this energy loss, the emission spectra of an excitedfluorophore is shifted to longer wavelengths when comparedto the excitation spectrum.

Ratiometric indicator compounds havea shift in either their excitation or emis-sion spectra on binding to ions. The exci-tation spectrum of radiometric dyes suchas the calcium dye Fura-2 changesaccording to the free Ca2+ concentration.The Ca2+ concentration is measured asthe ratio between two fluorescenceintensity values that are taken at twowavelengths.

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each time point, collect the data,and then use statistical analysis tobuild an overall picture of eventsoccurring over time.

However, live cell imaging pro-vides real-time images of cellularand subcellular events using mul-tiple molecular reporters over ex-tended periods of time and in threedimensions. To achieve live cellimaging, new tools have been dev-eloped that allow multiparametricanalysis while maintaining thefunctional viability of the cells. Mostlaboratories use completely auto-mated systems built around stan-dard epifluorescence microscopes,as opposed to laser scanning con-focal microscopes, and equippedwith temperature-controlled stageinserts to maintain 37˚C (whichpreserves the physiologic environ-ment).

The primary advantage of theepifluorescence scope is the sub-stantially lower level of illumina-tion required for imaging. Laserscanning confocal microscopy useslaser light to scan cells in a point-by-point fashion. Generally, theamount of light used in this tech-nique is high (particularly by in-experienced users) and leads toquenching or photodestruction ofthe fluorophore and phototoxicityto the cell.

This process may occur in partdue to excitation photons interact-ing with cellular components (e.g.,lysosomes or other granular light-absorbing structures) to generate

heat. More commonly, excitedfluorophores fail to transition backto ground state efficiently with theconcomitant release of the lower-energy photon.

Minimizing the frequency ofthese events is a major issue in livecell microscopy and entails a care-ful and judicious selection of fluo-rophore. Nevertheless, manage-ment of the excitation light budget,regardless of the fluorophore, is acritical issue in live cell imagingand can be enhanced by the use ofautomated shutters and filterwheels or monochromators, whichenable rapid switching of excita-tion wavelengths.

Various Fluorescent ProbesLabel Different

Subcellular StructuresWhile enormous amounts of infor-mation have been gleaned fromsimple cell migration or trackingexperiments, most live cell experi-ments typically combine transmit-ted light imaging with a fluorescentreporter tagged to either a subcel-lular structure (e.g., an organelle)or a cellular molecule (e.g., proteinor nucleic acid).

The labeling of subcellular orga-nelles can be achieved easily withmembrane-permeant, organelle-specific dyes. A host of dyes is com-mercially available, with a varietyof excitation and emission spectrathat can label the plasma mem-brane, nucleus, mitochondria, and

Ion-sensitive fluorophores allow moni-toring of intracellular parameters. In cul-tured mouse pulmonary artery endothe-lial cells, NO is shown to increase labilezinc. Endothelial cells were loaded withthe zinc-sensitive fluorophore FluoZin-3(Molecular Probes, Inc., Eugene, OR)and imaged at 37 °C using epifluores-cent microscopy. The pseudocoloredimages show the increase in fluores-cence over time in response to adminis-tration of the NO donor S-nitroso-cysteine ethyl ester and the rapid rever-sal of NO-induced increases in labilezinc by administration of the zinc chela-tor N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN).

BASELINE TPENNITRIC OXIDE5 MIN

NITRIC OXIDE10 MIN

CLAUDETTE M. ST. CROIXand BRUCE PITTare in the Department of Environ-mental and Occupational Health,University of Pittsburgh GraduateSchool of Public Health, and

SIMON C. WATKINSis in the Center for BiologicalImaging, Department of Cell Biol-ogy and Physiology, University ofPittsburgh School of Medicine,Pittsburgh, Pennsylvania.

[email protected]


other organelles. Functional dyesalso allow the fluorescent monitor-ing of intracellular parameters,such as pH, Ca2+ and other ion lev-els, and reactive oxygen and nitro-gen species.

One example of the use of ion-sensitive fluorophores in live cellimaging is provided in the figure,showing nitric oxide (NO)-induced,time-dependent increases in labilezinc in living primary cultures ofmouse pulmonary artery smoothmuscle cells, detected using thezinc-sensitive indicator FluoZin3(Molecular Probes, Inc.). The speci-ficity of this probe for zinc is shownby the decrease in fluorescencebelow baseline following applica-tion of the zinc chelator TPEN.

The labeling of individual mole-cules can be achieved with com-mercially available kits that react,for example, with free aminegroups. Such kits can be used tolabel proteins of interest directlyor, alternatively, to label primaryantibodies targeting a molecule ofinterest.

This technique is very usefulwhen directly examining cell-sur-face proteins or protein-proteininteractions at the cell surface. Forexample, we have used antibodiesconjugated directly to cell-surfacereceptors, as well as conjugatedligand, to study receptor internal-ization. However, the use of fluo-rescently conjugated molecules tostudy intracellular events typicallyrequires the use of invasive meth-ods, such as microinjection orscrape loading.

Genetically Encoded Fluorescent Probes Report

Protein BehaviorGenetically encoded fluorescentprobes are likely the most impor-tant development in optical imag-ing in the last decade. Most ofthese probes are based on greenfluorescent protein (GFP). GFP is aspontaneously fluorescent proteinoriginally isolated from the Pacificjellyfish, Aequorea victoria.

The fluorophore in GFP is deriv-

ed from three consecutive aminoacids: Ser65, Tyr66, and Gly67.Following synthesis of GFP, thesethree amino acids undergo auto-catalytic cyclization and oxidationto form the functional fluorophore.The fluorophore contains a seriesof conjugated double bonds thatprovide the fluorescent propertiesof GFP.

Several aspects of the GFP mol-ecule make it particularly attrac-tive for use in live cell microscopy.

• First, it is simple to make con-structs that contain GFP fused tothe genetic sequence of a proteinbeing studied. GFP consists of only238 amino acids (29 kD) and canbe fused to a variety of cellular pro-teins at either the COOH or NH2termini, without affecting the pro-tein’s native function or cellularlocalization.

• Second, GFP is highly stable,allowing its use in living cells aswell as in fixed or frozen cells andtissues.

• Finally, mutations in thethree amino acids comprising thefluorophore have produced GFPvariants with differing spectralcharacteristics, thus increasing thenumber of potential uses for GFP.

Green fluorescent protein (GFP),shown in a stereoview of its three-dimensional structure, consists of 11 β-strands (green ribbons) forming a hollowcylinder through which is threaded ahelix bearing the chromophore (yellow).

10 Å

FROM TSIEN RY: ANNU REV BIOCHEM 67:509-544, JULY 1998; WITH PERMISSION.


For example, a double mutationof Phe64→Leu and Ser65→Thryields enhanced GFP (EGFP), themost commonly used GFP variant.EGFP exhibits a red-shifted excita-tion maximum from 395 nm (wt) to488 nm, allowing for the use ofstandard fluorescein isothiocyanate(FITC) optics, and it is more photo-stable and brighter than the wild-type GFP.

GFP-based reporters have prov-ed to be invaluable tools to studycomplex biochemical processes inreal time, including the trackingand quantification of individual ormultiple proteins and the monitor-ing of protein–protein interactions.In addition, GFP-based indicatorshave been used as photo-modulat-able proteins to highlight and fol-low the fate of protein populationswithin a cell and as biosensors todescribe biological events and sig-nals.

Transmitted Light MicroscopyEnhances Capabilities ofFluorescence Techniques

In addition to fluorescent methods,transmitted light illumination isessential to any live cell imagingsystem. At a minimum, light micro-scopy is used only to validate thespecimen, but because live cellimaging does not allow contrast ofcells and tissues by traditionalstaining methods, other noncolori-metric brightfield-contrastingmethods must be used. However,these different methods may affectthe efficiency of fluorescent lightexcitation and detection of theemitted fluorescent signal.

Phase contrast microscopy de-pends on the presence of a pair ofphase plates: one in the condenserand the other in the objective. Asfluorescence imaging uses theobjective for transmission of both

DIFFRACTEDLIGHT

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Phase contrast microscopy depends on two specializedphase plates. Light waves passing through the condenserannulus illuminate the specimen and either pass undeviatedor are diffracted in phase by the cellular structures. The phaseplate in the objective then collects the undeviated and diffract-ed light to form the final image.Differential interference microscopy uses a Wollaston prismto create parallel wavefronts, which are differentially retardedby structures of different refractive indices in the specimen.When the wavefronts are recombined, destructive or construc-tive interference produces the final image.

Confocal microscopy uses a pinhole to focus the excitationlight (blue rays) onto a single point in the specimen. A secondpinhole aperture is placed in front of the detector, at a positionthat is confocal with the illuminating pinhole. This second pin-hole prevents light from regions outside the plane of focus(nonconfocal) from reaching the detector. A two-dimensionalimage of the precise plane of focus is created by scanning thebeam of light across the specimen using two oscillating mir-rors placed between the dichromic mirror and the objectivelens in such a way that the illuminating spotlight and confocalpinhole at the detector remain strictly in register.

GREG GAMBINO


the exciting light and emitted sig-nal, this phase plate can interferewith the signal recovered (up to30% of the available signal may belost). For this reason, this contrastmethod is generally avoided for anyinstrument trying to detect a low-level fluorescent signal. Phase con-trast is still useful when imagingcytoskeletal components in trans-mitted light, as these cellular fea-tures generally provide only mini-mal contrast on differential inter-ference contrast.

Differential interference contrast(DIC) is the preferred method forhigh-resolution transmitted lightimaging in live cell microscopy.DIC uses differences in the refrac-tive index of closely apposed struc-tures (e.g., at the edge of a cell, thelipid bilayer is adjacent to theaqueous media) to generate a high-resolution image. This techniqueallows exquisite resolution of cellu-lar motion.

DIC depends on polarized lightthat is then effectively split intoplane-parallel bundles using abirefringent material (e.g., calcite)within the microscope; this isknown as a Wollaston prism. Thelight then passes through the sam-ple, is recombined by a second Wol-laston prism, and lastly passesthrough a final polarizing filter,the analyzer.

Although the Wollaston prismhas little effect on light transmis-sion, the polarizing analyzer filtercan confound fluorescence signals(for both excitation and emission)considerably. However, it is possi-ble to remove the DIC analyzerduring fluorescence detection toavoid interference when it is notbeing used.

Motility assays are the mostbasic use of DIC imaging technolo-gy, used to visualize such things assperm motility and cell migration.

Perhaps the most significantaspect of DIC imaging and motilitymeasurements is that transmittedlight, rather than fluorescent emis-sion, is used as the collected signal.This illumination is low intensity,causing negligible phototoxicity to

the cell. In fact, microscopes forsimple cell motility assays can bequite simple, as the images may becollected at low light levels with noshutters or automation needed forfluorescent emission experiments.

In one recent motility experi-ment, we used DIC combined withGFP as a fluorescent label attach-ed to bacterial proteins to followdendritic cell uptake of bacteria.High-resolution live cell imaging ofEGFP-expressing Escherichia coliwas used to obtain a detailed pic-ture of particle uptake by dendriticcells derived from human peripher-al blood monocytes.

As the DIC techniques showed,immature dendritic cells undergopronouncedmorphologicchanges with-in minutes onexposure tononopsonizedE. coli. Thedendritic cellsdevelop ex-tensive mem-brane veilsthat efficientlycapture multi-ple bacteria.Internaliza-tion does notoccur in theveils, but in-stead, bacteriaare transport-ed to the cen-tral region ofthe cell, where they sink directlyinto the plasma membrane (seevideo 1).

Combining Contrast and GFP-Based Imaging Yields

Advanced TechniquesThe advent of GFP cloning tech-nologies has provided a means ofmaking specific proteins visible inliving cells, and one of the mostcommon applications of GFP inlive cell microscopy has been as alabel for specific proteins, allowingthe localization and trafficking ofthe protein in vitro. A number of

DIC techniques show uptake of liveEGFP-expressing E. coli by humanmonocyte-derived dendritic cells. Withinminutes of opsonization, the dendriticcells develop extensive morphologicveils that capture other bacteria.¸ See video 1 associated with thispaper at www.sciandmed.com/sm/.

SALTER RD, ET AL: J LEUKOCYTE BIOL 75:240-243, FEB 2004.

DENDRITICCELL

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sophisticated methods have beendeveloped to study molecular be-havior and interactions with highspatial and temporal resolution.

One of the earliest methodsused to investigate steady-stateprotein dynamics was fluorescencephotobleaching, in which a fluoro-phore is made nonfluorescent byexposure to high-intensity light.The bleached region serves as areference mark to track labeledproteins. Derivations of photo-bleaching methods include fluores-cence recovery after photobleach-ing (FRAP), fluorescence loss inphotobleaching (FLIP), and fluo-rescence localization after photo-bleaching (FLAP).

Various other fluorescence-basedtechnologies are useful for specificapplications, but the two techni-ques used most commonly in ourlaboratories are fluorescence reso-nance energy transfer (FRET) andtotal internal reflection fluores-cence (TIRF).

Fluorescence Resonance Energy Transfer (FRET)

The development of GFP variantswith differing absorbance andemission spectra has allowed forthe simultaneous visualization ofmultiple tagged molecules within acell. In particular, the pairing ofthe cyan (CFP) and yellow (YFP)fluorescent proteins has been cru-cial in advancing the use of FRETto study inter- and intramolecularinteractions in living cells.

FRET is a quantum mechanicalprocess whereby two fluorophores(i.e., a donor and an acceptor) thathave appropriate spectral proper-ties and are closely apposed (10-50Å) transfer photon energy in a non-radiative fashion. The FRET effectdecreases rapidly as the distancebetween the donor and acceptorfluorophores increases.

Two general strategies haveevolved using GFP variants inFRET-based applications. The firstinvolves the incorporation of GFPdonor and acceptor fluorophores atopposite ends of a conformationallyactive protein. Alterations in pro-tein conformation change the rela-tive positions of the two fluoro-phores, and this change is reflectedin alterations in FRET.

The second strategy involves theencoding of GFP donor and accep-tor fluorophores on two distinct pro-teins. In this case, FRET betweenthe two fluorophores provides evi-dence for a physical interactionbetween the two proteins. Thismethod has been used widely inliving cells to support in vitro dataprovided by immunoprecipitationand other biochemical techniques.

Verification of the occurrence ofFRET, in both inter- and intramol-ecular applications, is obtained byusing selective acceptor photo-bleaching to generate an increasein donor fluorescence. A secondcontrol for demonstrating inter-molecular FRET is to show thatbiologically inert mutants of the

Fluorescence resonance energytransfer (FRET). The donor fluorophore(cyan) is excited by light of a specificwavelength. The excited state energy istransferred nonradiatively to the acceptorfluorophore (yellow ) which then emitslight at its characteristic wavelength.In one FRET-based application (inset ),donor and acceptor GFP fluorophoresare incorporated at opposite ends of aprotein. As protein conformationchanges, the relative positions of thetwo fluorophores are altered, causing adetectable change in emissions of boththe donor and acceptor.

CFP

λem=525 nmλem=480 nm

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GREG GAMBINO


interacting proteins do not under-go FRET.

Methods for detecting FRETbetween a donor and an acceptorwith overlapping excitation andemission spectra require the use ofnarrow detection bands and auto-matic switching of optical filters todifferentiate between emissions,along with complex mathematicalcorrections to account for crosstalkbetween channels.

Recent advances in detectortechnology, however, now enablethe resolution of fluorescent im-ages, providing full spectral infor-mation for each voxel of the imagewithout the need for switchingoptical filters. This method usesconfocal imaging and is one situa-tion in which confocal methods maybe more appropriate than widefieldimaging for live cell studies. Withconfocal methods, it is easy toselectively photobleach acceptormolecules, and by using calibrationspectra, the cross-talk betweenoverlapping cyan and yellow emis-sions can be separated. The use ofconfocal methods allows the detec-tion of small, but potentially bio-logically meaningful, changes inFRET that are common with gen-

etically encoded reporters and thatare extremely difficult to resolvereliably using more traditionalmethods based on bandpass filters.

We used confocal-based spectralimaging to examine the role ofmetallothioneins (MT) in nitricoxide signaling. Previous data hadsuggested that metal ion homeo-stasis is regulated in part by a linkbetween MT and cellular redoxstatus via the interaction of MTwith the free radical NO.

We used a chimera reporterfusion protein consisting of MTsandwiched between enhancedCFP (ECFP) and enhanced YFP(EYFP) molecules, and FRET be-tween ECFP and EYFP was usedto monitor conformational alter-ations in the core MT protein.Alterations in the FRET-MT sig-nal, indicative of metal release,were produced by exposing live,FRET-MT-expressing pulmonaryendothelial cells to NO donors oragents that activate intracellularNO synthesis.

The GFP-FRET method alsohas been used to study a variety ofother intracellular signaling path-ways using reporter molecules forcalcium, cGMP, cAMP, and tyro-

FRET-based detection of metallothion-ein-NO interaction. A FRET-MT reporterwas constructed of human type IIa met-allothionein (MT), flanked by ECFP andEYFP molecules. MT unfolding, inducedby metal chelators or nitric oxide (NO),results in metal (zinc) release and con-sequent changes in FRET.In this experiment, pulmonary endothe-lial cells were infected with an adeno-viral vector encoding the fluorescentFRET-MT reporter molecule and imag-ed 24 hours later. FRET was detected inreal time, using full spectral confocalimaging. The graph shows the separa-tion of the two emitted signals (cyan andyellow) following spectral unmixingbased on individual calibration spectrafor each protein.The spectral report (far right ) shows anincrease in the peak of the donor (cyan)and decrease in that of acceptor (yel-low) following a 5-min exposure to anNO donor.

FRET

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FROM ST. CROIX CM, ET AL: FREE RADIC BIOL MED37:785-792, 15 SEP 2004; WITH PERMISSION.

BASELINE NITROSOTHIOL

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sine kinase activity, among others. A potentially powerful way to

detect FRET is by combiningFRET with a second advancedimaging modality known as fluo-rescence lifetime imaging micro-scopy (FLIM). FLIM measures theextent to which a donor’s fluores-cence lifetime is shortened byFRET events, using a pulsed laserto excite the probes and a gatedcamera to detect the fluorescenceof the donor.

The use of FLIM to measureFRET efficiency is particularlyuseful when there is significantoverlap of the donor fluorescenceand the acceptor emission wave-lengths or when the donor is proneto photobleaching. CombiningFRET and FLIM technologies,however, requires highly sophisti-cated equipment and a great dealof technical expertise.

Total Internal Reflection Fluorescence Microscopy (TIRF)

The physical phenomenon of totalinternal reflection relies on therefraction (bending) of light as itencounters the edge between twomedia with different refractive in-dexes, resulting in the confinementof some or all of the light to thehigher index medium. In TIRF,light from laser sources is directedat a specimen on a glass coverslipin such a way as to create an evan-escent wave propagating parallelto the interface between the cover-

slip and the aqueous medium dueto nearfield effects. The intensity ofsuch a wave falls off exponentiallywith distance from the coverslip,resulting in excitation of only thefluorophore at the portion of thecell nearest the coverslip (therebyestablishing optical slices of <100nm).

As no photons are used for illu-mination, this method not onlyprovides emission from a limited,defined focal volume but also offersthe highest signal-to-noise imagecurrently possible in light micro-scopy. In addition, with carefulcontrol of light intensity, photo-bleaching and phototoxicity arereduced.

The basic principles of TIRFwere described in the early 1980s,but the recent development of im-proved objectives, sensitive detec-tors, and genetically encoded GFP-based reporters has led to a resur-gence of interest in its application.TIRF is currently used, either byitself or in combination with otheroptical approaches, to study vari-ous physiologic events associatedwith membrane trafficking, vesicu-lar transport, secretion, and exocy-tosis. The growing availability ofmulticolored TIRF systems willgreatly increase the number andcomplexity of molecular questionsto be resolved with this technology.

We used simultaneous TIRF tocharacterize the dynamics andinteractions of components of the

Total internal reflection fluorescencemicroscopy (TIRF). When an excitationlight beam traveling at a low anglethrough a dense medium (e.g., glasscoverslip) reaches an interface with lessdense medium (e.g., aqueous solution,cell surface), it causes all of the light tobe reflected at the cell surface ratherthan passing through the cell. Thereflection creates a thin evanescent field100 to 200 nm deep. Fluorophores nearthe cell surface or interface are excitedby the evanescent wave, but fluoro-phores farther away are not. This effectproduces a high-contrast image ofevents occurring at or near the cell sur-face.

COVER SLIP

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endocytotic machinery. In eukary-otic cells, a clathrin coat forms onmembranes as a polygonal lattice,which progressively curves to in-corporate membrane, cargo, andextracellular fluid into a transportvesicle. The kinetics of clathrincoat assembly and the mechanicsof adaptor protein interaction arenot well understood.

We used TIRF microscopy toextend the time-resolved study ofclathrin structures by analyzingthe temporal co-localization ofEGFP-clathrin with fluorescentlylabeled, compartment-specificadaptor proteins and/or cargo mol-ecules. We showed that most cla-thrin structures are relatively stat-ic, moving vertically in and out ofthe evanescent field, but with littlelateral motion. A small minority ofclathrin-positive puncta, however,was distinct from plasma mem-brane clathrin lattices; these werehighly motile and associated withendocytosed cargo molecules, re-vealing an endosomal populationof clathrin structures (see video 2).

Intravital Imaging PermitsStudy of Intact Living Tissue

Some established experimentalmodels have been adapted to allowfor the direct fluorescent imagingof intracellular events in intact tis-sues. In particular, the ability to

isolate and perfuse the circulatorysystems of either the heart or lungsof rodents has enabled researchersto use a variety of cell-permanentfluorescent probes, including thosesensitive to ion flux, redox status,and pH, to study the integrativenature of signaling events at boththe subcellular and intercellularlevels.

In addition, fluorescent imagingtechniques have been used to studythe vascularization of organs andtheir regulation of blood flow, in-cluding measurement of the distri-bution and movement of fluores-cent beads or indicators present inthe perfusate. In fact, in situ imag-ing has always been at the fore-front of contemporary pulmonaryresearch, ever since video-micro-scopy was used to determine pul-monary capillary transit time bymeasuring the time required forfluorescent dye to pass from anarteriole to a venule on the surfaceof an intact dog lung.

More recently, both epifluores-cent and confocal microscopy tech-niques have been used to imageisolated, perfused rat lung. Thesestudies were intended to: 1) observeintercellular calcium signaling atthe alveolar-capillary junction; 2)study the role of reactive oxygenspecies (ROS) in ischemia/reperfu-sion injury in lung tissue; and 3)measure transalveolar transport of

ADAPTED FROM KEYEL PA, ET AL: J BIOL CHEM 279(13):240-243, 26 MAR 2004.

Clathrin dynamics in living cells werevisualized with TIRF microscopy. Theleft panel shows a single human fibro-blast expressing GFP-labeled clathrinlight chain α (GFP-LCα). Motion withinthe boxed region is shown by color cod-ing of GFP-CLα in the right panel, withwhite representing stationary objects. ¸ See video 2 associated with thispaper at www.sciandmed.com/sm/.

water, protons, and solutes by add-ing membrane-impermeant fluoro-phores to the airspace fluid.

The use of specific promotersand targeting signals to introduceGFP-tagged proteins into theintact organism and to direct theproteins to specific tissue regions,cell types, and subcellular com-partments has provided spatiotem-poral information in complex tis-sues, thereby placing it in a physio-logic context.

The utility of this approach wasdemonstrated in an experimentusing confocal-based imaging tostudy surface vasculature in theisolated perfused lung of a TIE2-GFP mouse. This mouse expresses

GFP under the direction of theendothelial-specific receptor tyro-sine kinase (Tie2) promoter. Usingsequential XYZ-sections, the three-dimensional anatomy of the vascu-lature was reconstructed, and realmeasures of cross-sectional areaand maximal diameter were gener-ated. This application is potentiallyuseful for measuring vascular reac-tivity in response to either inhaledor circulating agonists.

Significant advances in genetransfer technologies in recentyears also permit the introductionof genetically encoded probes intointact animals to study physiologicsignaling events in their appropri-ate microenvironment. For exam-

Laser scanning confocal microscopy of the sub-pleural vasculature of a perfused lung isolated from aTie2-GFP mouse. The TIE2-GFP mouse (JacksonLabs.) expresses GFP under the direction of theendothelial-specific receptor tyrosine kinase (Tie2) pro-moter and thus can be used to visualize pulmonary ves-sels. Using sequential XYZ-sections (512 X 512 pixels,at Nyquist axial frequency 250 nm) which include theentire vessel being studied, it was possible to recon-struct the vessel and generate real measures of cross-sectional area and maximal diameter using commercial-ly available software. The white bars mark the diame-ters of small pulmonary vessels.


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Multiphoton laser scanning micro-scopy of deep tissue uses two long-wavelength (near-IR) photons to gen-erate the same electronic transition asa single shorter wavelength photon.


ple, endothelial expression of theFRET-MT reporter in mouse lungwas achieved using injections ofDOTAP:cholesterol liposomes fol-lowed by adenovirus containingcDNA for FRET-MT. Spectral laserscanning confocal imaging of thesubpleural vasculature of the iso-lated perfused mouse lung confirm-ed that NO donors caused confor-mational changes in the FRET-MTreporter in the intact pulmonaryendothelium, as previously shownin live cultures of endothelial cellsdescribed earlier.

The imaging of intact tissue canprovide information about the

spatial distribution of biologicalphenomenon over time within aparticular organ, provided that thechosen technology has the appro-priate depth penetration and theability to perform computationalthree-dimensional reconstructionof images after acquisition. As withlive cell microscopy, maintainingthe viability of the tissue is also aconcern during serial imaging.

Confocal approaches are gener-ally limited by the scattering andabsorbance of light entering the

tissue. This limits the localizationof excitation, while scattering ofthe emitted photons by the tissuecauses much of the emitted signalto be lost. To successfully imagedeep within light-scattering mater-ial, it is necessary to use a long-wavelength illumination systemtogether with a detection path thatis minimally affected by scattering.

Although some of the limita-tions of short-wavelength scatter-ing (e.g., for blue light at 488 nm)by living tissue can be overcome byoperating in the near-infrared re-gion of the spectrum, this approachposes its own limitations.Currentlyonly a few near-infrared-excitablefluorescent dyes are available forapplication in cellular imaging,and although the longer emissionwavelengths are less prone to scat-tering, the detection of emittedphotons is still subject to projectionthrough a detection pinhole toreject out-of-focus signal. Thus,multiphoton imaging has becomethe method of choice for thesetypes of studies.

Multiphoton laser scanningmicroscopy (MPLSM) relies on thesimultaneous absorption of two

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FRET detection in the isolated perfused mouse lung. Ex-pression of the FRET-MT reporter in pulmonary endotheliumwas archieved via injection of DOTAP:cholesterol liposomesfollowed by adenovirus containing cDNA for FRET-MT.A, Confocal imaging of a section of murine lung tissue 48 hrsafter injection shows co-localization of staining for endothelial-specific platelet/endothelial cell adhesion molecule (PECAM,red) and FRET-MT expression (yellow), suggesting expressionis predominantly endothelial. DAPI (4' ,6-diamidino-2-phenyl-indole) binds to dsDNA and labels nuclei of all cell types.

B, Confocal imaging of the isolated mouse lung, perfused withlabeled albumin (magenta), shows the vascular network,whereas FRET-MT expression (yellow) is largely confined tothe microvasculature. C, Spectral laser scanning confocal imaging of the subpleuralvasculature of the isolated perfused mouse lung shows thatthe addition of the NO donor DETA-nonoate to the perfusatecauses an increase in the peak emission intensity of the donor(cyan ~485 nm) and a decrease in that of the acceptor (yellow525 nm), consistent with conformational changes in FRET-MT.

A B C


long-wavelength (near-infrared)photons to generate an electrontransition within a fluorophoreequivalent to that produced by asingle high-energy photon. The dif-fraction-limited focusing and

pulsed nature of the lasers usedresult in an excitation event onlyat the plane of focus of the illumi-nating lens, avoiding the need for apinhole aperture. Also, there is lessphototoxicity.

Multiphoton laser scanning micro-scopy shows a high-resolution three-dimensional image of the distal conduct-ing airways of the lung. The red fluores-cence marks cells that are immuno-reactive for Clara cell secretory product.

In vivo imaging of insulin-Timer islets transplanted underthe renal capsule of a mouse and accessed via a transdermalwindow affixed to the flank. On multiphoton laser scanningmicroscopy (MPLSM, right ), the timer protein changes fromgreen to red over the first 24 hours after synthesis and there-fore can be used to monitor the rate of insulin production inimages taken over an extended time period. At the single time

point shown, pockets of newly synthesized insulin (greenarrow) and areas of more mature protein (yellow to red) areseen within the same field of islets.


Intravital Imaging Enables In Situ Visualization in Intact Living Animals

The ultimate goal of biomedicalresearch is to identify, follow, andquantify biological processes at thecellular and subcellular level in theintact organism. One means ofachieving this goal has been withthe development of chronic animalmodels incorporating transparentwindows, enabling the visualiza-tion of temporal physiologic eventsusing intravital microscopy. Thesetechniques, in combination withsurgical implantation of GFP-transduced cancer cell lines intometastatic rodent models, havebeen particularly useful in thestudy of tumor vascular functionand tumor-host interactions.

As in deep tissue imaging, theadvantages of multiphoton laserscanning microscopy (MPLSM) foracquiring three-dimensional data,while limiting light scattering andphototoxicity, are particularly suit-ed for high-resolution in situ imag-ing. The value of combining ad-vances in transgenic technologieswith increasingly sophisticatedimaging modalities, to addressphysiologically relevant questions,was illustrated by the developmentof a transgenic mouse expressingproinsulin II tagged with the fluo-rescent reporter protein, Timer, toinvestigate insulin secretion.

Timer is a mutated version of ared fluorescent protein from theAnthozoa coral. The mutations per-mit its fluorescence to change from

green, when it is first synthesized,to red over a period of about 24 hrs.The age of the protein tagged withTimer can be determined by theobserved ratio of green to red fluo-rescence. Pancreatic β-cells inthese mice fluorescently revealwhen they are producing insulinand how long it has been sincethey synthesized it.

Transplantation of Ins-C-Timerislets under the renal capsule haspermitted the in vivo visualization,by MPLSM, of insulin synthesisthrough a body window deviceimplanted over the transplant site.The window technique allows formulticolor imaging, over a periodof several days, of both insulin-pro-ducing cells and T cells labeledwith a fluorophore distinguishablefrom Timer. This model may proveuseful in investigating the etiologyof type 1 diabetes, as well as inmonitoring the success of therapiesbased on allotransplantation ofislet cells.

The increasing versatility ofimaging probes, coupled with

dramatic improvements in instru-mentation and analysis tools, haveprovided important insights intomyriad aspects of biological func-tion at the subcellular, cellular, tis-sue, and organismic level. The pri-mary challenges ahead involve thetranslation of these new tools andthe knowledge base they havespawned in the understanding ofbiological responses to environ-mental variables.

RECENT REVIEWSDerek Toomre, Dietmar J. Manstein: Lighting up the cell surface with

evanescent wave microscopy. Trends Cell Biol 11(7):298-303, 1 Jul2001.

Cornelis J. Weijer: Visualizing signals moving in cells. Science300(5616):96-100, 4 Apr 2003.

Warren R. Zipfel, Rebecca M. Williams, Watt W. Webb: Nonlinear magic:multiphoton microscopy in the biosciences. Nat Biotechnol 21(11):1369-1377, Nov 2003.

Jennifer Lippincott-Schwartz, George H. Patterson: Development anduse of fluorescent protein markers in living cells. Science 300(5616):87-91, 4 Apr 2003.

Roger Y. Tsien: The green fluorescent protein. Annu Rev Biochem 67:509-544, July 1998.

ORIGINAL PAPERSSuzanne Bertera, X. Geng, Z. Tawadrous, et al: Body window-enabled in

vivo multicolor imaging of transplanted mouse islets expressing aninsulin-Timer fusion protein. Biotechniques 35(4):718-722, Oct 2003.

Peter A. Keyel, Simon C. Watkins, and Linton M. Traub: Endocytic adap-tor molecules reveal an endosomal population of clathrin by total inter-nal reflection fluorescence microscopy. J Biol Chem 279(13):13190-13204, 26 Mar 2004.

Russell D. Salter, Renee J. Tuma-Warrino, Paul Q. Hu, Simon C. Watkins:Rapid and extensive membrane reorganization by dendritic cells follow-ing exposure to bacteria revealed by high-resolution imaging. J LeukocBiol 75(2):240-243, Feb 2004.

Claudette M. St. Croix, Molly S. Stitt, Karanee Leelavanichkul, et al:Nitric oxide-induced modification of protein thiolate clusters as deter-mined by spectral fluorescence resonance energy transfer in liveendothelial cells. Free Radic Biol Med 37(6):785-792, 15 Sep 2004.

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Imaging of Biological Systems with Fluorescent Technologies