Single-Molecule Fluorescence Resonance Energy Transfer
SSingle-Molecule FluorescenceResonance Energy TransferAlexander E. Johnson-Buck1, Mario R. Blanco2and Nils G. Walter11Department of Chemistry, The University ofMichigan, Ann Arbor, MI, USA2Department of Cellular and Molecular Biology,The University of Michigan, Ann ArborMI, USASynonymsFluorescence resonance energy transfer (FRET);Single-molecule Förster resonance energy transfer; Single-pair fluorescence resonance energytransferDefinitionSingle-molecule fluorescence resonance energytransfer (smFRET) is a technique used to measurenanometer-scale distances between specific siteson an individual molecule, usually as a functionof time.IntroductionIn this entry, we review smFRET, a powerfultechnique for measuring distances and monitoringdynamics at the molecular scale. In this technique,the researcher monitors distances between two ormore individual fluorescent labels by measuringhow efficiently electronic energy is transferredbetween them, a phenomenon known as Försterresonance energy transfer (FRET). Its power liesin its ability to detect distance changes as small as 0.3 nm in individual molecules (or pairs of molecules) within a heterogeneous population (Royet al. 2008). Although it requires a specializedfluorescence microscope, smFRET is employedin many laboratories worldwide and has beenused to address questions about topics rangingfrom intermolecular interactions to macromolecular folding and catalysis.Although smFRET has only been technicallypossible since the mid-1990s, its theoretical foundations were laid much earlier through thepioneering work of Theodor Förster in the 1940sand through others who elucidated the distancedependence of FRET and its use as a molecularruler (Stryer 1978). A separate branch of inquiry,the first single-molecule measurements of ionchannels using the patch clamp technique, alreadyestablished some of the core aspects of analyzingtime-lapsed recordings from individual moleculesin the 1970s and early 1980s (Sakmann and Neher2009). By the mid-1990s, improvements in fluorescence detection brought about by total internalreflection fluorescence microscopy made it possible to measure the weak emission of single organicfluorophores under ambient conditions and, soonthereafter, smFRET was realized (Ha et al. 1996).# European Biophysical Societies’ Association (EBSA) 2018G. C. K. Roberts, A. Watts (eds.), Encyclopedia of -9 492-1
2Single-Molecule Fluorescence Resonance Energy TransferEBETBhvhvThvGGDonorAcceptorSingle-Molecule Fluorescence Resonance EnergyTransfer, Fig. 1 Simplified electronic diagram of FRET.Solid lines represent rapid electronic transitions, anddashed lines represent slow transitions. A FRET donorcan be excited from its ground state (G) to an excitedstate (E) by a photon of energy hn (blue). The exciteddonor can then return to the ground state, emitting a photonof lower energy (i.e., it can fluoresce, green); enter anonfluorescent triplet state (T); permanently photobleach(B); or donate its energy to a nearby acceptor molecule byFRET. The excited acceptor can then similarly emit aphoton (red), enter a triplet state, or photobleachWe begin with a discussion of the theory ofFRET as it applies to single-molecule experiments, followed by a brief description of a typicalsmFRET experiment, including the necessaryequipment and materials. We then discuss someof the major insights gained from smFRET, aswell as some of its strengths and limitations.Finally, we conclude by commenting on recentand ongoing developments in the field.For FRET to occur efficiently, the fluorescenceemission spectrum of the donor must overlap considerably with the absorption spectrum of theacceptor, i.e., the fluorophores must be in resonance (Fig. 2), the two molecules must be within acertain distance of one another (typically 10 nm), and their transition dipole momentsmust be in (partial) alignment. At the same time,their absorption spectra should be separateenough that the donor can be excited with highspecificity, and their emission spectra sufficientlyseparate to ensure specific detection of both donorand acceptor.Since FRET is a dipole–dipole interaction, itsefficiency E depends on the sixth power of theseparation r between the donor and acceptor as:Photophysical Basis of smFRETIn 1948, Förster developed the theory governingthe non-radiative transfer of energy from oneelectronically excited molecule to another nearbymolecule. When a fluorescent molecule(fluorophore) is excited by a photon of the properenergy, it returns to the ground state via one ofseveral possible pathways: It can dissipate itsenergy by emitting a photon, by transferringenergy to other molecules without emitting a photon (non-radiatively), or by reacting chemically(Fig. 1). In FRET, one form of non-radiativeenergy transfer, the excited “donor” fluorophoretransfers its excitation energy to a nearby “acceptor” fluorophore through an electronicdipole–dipole interaction. The excited acceptormay then return to its ground state via one ofvarious pathways, including by emitting a photon(fluorescence).E ¼ r 6 r 6 þ R 60 (1)where the Förster radius R0, corresponding to theseparation at which energy transfer is 50% efficient, is:R0 ¼ J k2 Q0 n 4 1 6 9:7 103 A(2)The value E, which can be calculated from therelative fluorescence of the donor and acceptor(see “Data Processing and Analysis,” below),thus reports on the distance between the twofluorophores. The value of R0 depends on theoverlap integral J between the donor emission
Single-Molecule Fluorescence Resonance Energy Transfer3AcceptorDEAbsorbance / e Fluorescence Resonance EnergyTransfer, Fig. 2 Desirable spectral properties for aFRET donor–acceptor pair. For specific fluorescent excitation and detection, the absorption spectra (solid curves)of the donor and acceptor should overlap little, as shouldtheir emission spectra (dashed curves). However, for efficient FRET to occur, the emission spectrum of the donorshould overlap considerably with the absorption spectrumof the acceptorand acceptor excitation spectra, the so-called orientation factor k2 describing the relative orientations of the donor and acceptor, the quantum yieldQ0 of donor fluorescence in the absence of acceptor, and the refractive index n of the medium inwhich the interaction takes place. If thefluorophores are freely rotating at a rate fasterthan the excited state lifetime of the donor,k2 2/3, and R0 is constant for a givendonor–acceptor pair in aqueous solution. Thiscan be confirmed for a particular system by measuring the fluorescence anisotropy of bothfluorophores.Experimental Design and DataAcquisitionIn order to use smFRET to measure conformational changes or interactions between molecules,the molecule(s) of interest are labeled, usually atspecific sites, with donor and acceptorfluorophores that report on a distance of interest.The ideal fluorophore is stable under high photonflux, has high molar absorptivity and fluorescencequantum yield, and undergoes minimal“blinking” (spontaneous excursions into0R0distance2R0Single-Molecule Fluorescence Resonance EnergyTransfer, Fig. 3 Distance dependence of FRET. Efficiency of energy transfer (E) is a steep function of distanceat values near R0nonfluorescent states). The fluorophores used aregenerally small ( 1 nm) organic molecules, thetwo most common being the cyanine dyes Cy3and Cy5, though the Alexa Fluor and ATTO seriesof dyes appear comparable. Quantum dots andfluorescent proteins can also be used in smFRET,but their use has been more difficult to implementbecause they are larger and, in the case of fluorescent proteins, less photostable (Roy et al. 2008).Positions of the fluorophores should ideally bechosen such that the distance r between them isclose to the Förster radius since that is whereFRET efficiency is most sensitive to changes inr (Fig. 3).All organic fluorophores eventually photobleach, permanently losing their fluorescenceproperties through reaction with molecular oxygen (Figs. 1 and 4). This is useful because theinstantaneous loss of fluorescence signal uponbleaching is evidence that the fluorescence originates from a single molecule. However, becausephotobleaching also reduces the time window ofobservation, it is often desirable to delay it asmuch as possible. So-called oxygen scavengingsystems reduce the concentration of oxygen insolution by catalyzing its reaction with substratesother than the FRET donor and acceptor. The mostcommon systems use either: (1) the enzymes glucose oxidase and catalase in combination with thesubstrate glucose or (2) the enzyme
Single-Molecule Fluorescence Resonance Energy 0100150200250300TimeSingle-Molecule Fluorescence Resonance EnergyTransfer, Fig. 4 Simulated typical FRET time trace of asingle molecule. The fluorescence intensity counts of thedonor and acceptor change in a discrete, anticorrelatedfashion, reporting on underlying molecular distancechanges that are detected as transitions between high andlow FRET efficiency (Eapp). Upon photobleaching, thedonor and acceptor intensity counts instantaneously fallto 0, evidence that the signal originated from a singleFRET pairprotocatechuate dehydrogenase and its substrate,protocatechuic acid. A related problem isblinking, a term used for temporary nonfluorescence caused by excursions to kineticallytrapped triplet states (Fig. 1). When oxygen, agood triplet state quencher, is removed from solution using a scavenger system, other additives(Trolox, b-mercaptoethanol) are often employedto dramatically reduce blinking.Observation of the weak fluorescence signalfrom single molecules requires: (1) high-poweredillumination, (2) means of reducing or rejectingbackground fluorescence that would otherwisegreatly diminish the signal-to-noise ratio, and(3) sensitive detection. To meet requirement (1),the illumination is almost always provided by thehigh-powered, monochromatic light of lasers.Requirement (2), the reduction of backgroundfluorescence, is usually achieved by excitingonly a small volume of the sample by means ofone of the following illumination schemes: totalinternal reflection fluorescence (TIRF), confocal,highly inclined, and laminated optical sheet(HILO), near-field scanning optical (NSOM), orzero-mode waveguides (Walter et al. 2008). Mostcommon of these illumination schemes is totalinternal reflection fluorescence (TIRF), whichreduces background fluorescence by illuminatingonly that part of the sample that is within 100 nmof the surface of the microscope slide or coverslip.This requires immobilizing the molecules of interest at the illuminated interface, which is usuallyachieved using specific, high-affinity bindingsuch as the streptavidin–biotin interaction. Toresolve single molecules, they must beimmobilized from a very dilute ( 100 pM) solution, resulting in a surface density no larger thanabout 0.2 mm 2. Surface immobilization has theadded benefit of allowing one to observe the samemolecule over several seconds, minutes, or evenhours. Finally, requirement (3), sensitive detection, is usually provided by an electron multiplying charge-coupled device (EMCCD) camera inthe case of wide-field illumination, such as TIRF,or avalanche photodiodes in the case of pointdetection schemes like confocal microscopy.An smFRET experiment also requires opticsfor filtering out stray excitation light and directingfluorescence from the donor and acceptor intoseparate detection channels. Scattered excitationlight is removed from the detection path usinghigh-optical density filters that only transmit certain frequency bands. Separation of donor andacceptor signals is accomplished using dichromatic (dichroic) mirrors, which reflect specificfrequencies of light and transmit others.
Single-Molecule Fluorescence Resonance Energy TransferAdditional mirrors are then used to direct theemission signal onto the detector(s) so that thedonor and acceptor emission can be measuredsimultaneously.Data Processing and AnalysisProcessing of smFRET data from raw cameramovies involves: (1) locating and matchingcorresponding donor and acceptor signals in thefield of view, (2) matching each donor signal withits acceptor signal (channel registration), and(3) determining the FRET efficiency as a functionof time for each molecule throughout the movie(Fig. 4). Since the donor and acceptor channels ofthe CCD image do not typically have a linearcorrespondence, they are generally mappedusing a higher-order polynomial transformationto ensure correct assignment of donor–acceptorpairs. Usually so-called fiduciary markers (suchas fluorescent beads) that are visible in both channels are used to establish this mapping.The apparent FRET efficiency Eapp can becalculated as:Eapp ¼IAIA þ ID g(3)where ID and IA are the total number of photonsemitted per movie frame by the donor and acceptor, respectively. The parameter g, which dependson the relative quantum yields and detection efficiencies of the donor and acceptor, can be calcuDI Alated as g ¼ DI, where DIA and DID are theDchanges in intensity of the acceptor and donorintensity upon photobleaching of the acceptor. Ifthe Förster radius is known and if anisotropy ofboth fluorophores can be ruled out (see “Insights,Strengths, and Limitations,” below), Eapp can berelated to absolute distances according to Eq. (1).If not, Eapp still gives an estimate as to the relativedistances between donor and acceptor in differentFRET states (Roy et al. 2008).Analysis of the Eapp versus time data variesgreatly depending on the behavior of the sample.The typically low signal-to-noise ratios of single-5molecule detection often make it challenging todetermine the number and values of differentFRET states in a given molecule. To facilitatethe assignment of FRET states, hidden Markovmodeling (HMM) or nonlinear filters may beapplied. Histograms of FRET efficiency are usually generated from the time traces of hundreds ofmolecules under the same conditions and canoften be fit with multiple Gaussian distributionsto estimate the number of states and their values ofEapp (Fig. 5), thus providing information about theequilibrium properties of the system (Roy et al.2008).If the molecules show transitions between discrete Eapp values over time (i.e., dynamics), eitherHMM or analysis of the time spent in each state(dwell times) can be used to estimate the underlying kinetics of the system (Fig. 5). Genuine FRETtransitions are characterized by inversely proportional changes in acceptor and donor signal intensity, or anticorrelation (Figs. 4 and 5), a propertythat can be used to filter out spurious transitionsdue to other photophysical phenomena such aschanges in the local environment of one of thefluorophores. Transition density plots, whichsimultaneously display the probability of all possible transitions between different FRET states,are often compiled based on HMM to provide aglobal view of the observed dynamics. If certaintransitions have slow kinetics or appear onlyinfrequently in time traces, evidence of themmay be suppressed by faster (more frequent) transitions; in this case, the probability that a transition occurs at least once within a given moleculemay instead be plotted (Blanco and Walter 2010).Insights, Strengths, and LimitationsSingle-molecule FRET has several strengths.First, the steep distance dependence of Eapp nearR0 and specificity of the energy transfer betweendonor and acceptor make it possible to monitorvery specific interactions and events. Second, theratiometric property of smFRET renders it one ofthe most sensitive and robust single-moleculetechniques. Third, when using appropriate oxygenscavengers,blinkingsuppressants,and
tor1200100080060040020000.0–0.20.8––0.590.37 0.540.50.8 0.46 0.710.5 1.180.2IsFsFs1.000.20.40.60.81.0Is0.2 0.4 0.6 0.8 1.0Transition Density Plot0.5EappFRET histogramAggregate AnalysisRate Constants (s-1)Counts14012010080604020function of time (middle). An aggregate analysis of typically hundreds of moleculesyields thermodynamic information, such as a histogram of the population distribution ofthe various FRET states, as well as first-order rate constants and a probability plot oftransitions from each FRET state (IS) to each other state (FS) (right)FRET TrajectoriesSingle-Molecule Fluorescence Resonance Energy Transfer, Fig. 5 Schematicworkflow of smFRET analysis for a simulated three-state system. Each acceptor (oneof which is encircled in red) is paired with its corresponding donor molecule (circled ingreen) in the CCD image (left). Using the intensity of donor and acceptor in each movieframe, the apparent FRET efficiency Eapp is calculated in each frame and plotted as aEappPairing Donorswith Acceptors6Single-Molecule Fluorescence Resonance Energy Transfer
Single-Molecule Fluorescence Resonance Energy Transferacquisition hardware, smFRET can be used tomonitor molecular events occurring on timescalesranging from 1 ms to hours. Because of theseproperties, smFRET is particularly well suited tomonitoring conformational changes in singleimmobilized molecules over long periods oftime, but has been used to study phenomena ranging from the dynamics of motor proteins and RNAenzymes to structural transitions in DNA .Perhaps the greatest advantage of smFRET, aswith “Single-Molecule Spectroscopy” in general, lies in its ability to discern heterogeneousbehavior within a population of molecules at equilibrium without the need for rapid mixing or othersynchronization. In studies of conformationaldynamics in protein and RNA enzymes, smFREThas repeatedly revealed heterogeneous behavior,that is, the kinetics of conformational transitionsvaries over time or from molecule-to-molecule.Some molecules even appear to have multiplenative states with the same activity but subtlydifferent structures that do not interconvert. Thisstands in contrast with the classical view of macromolecules as having a unique native state with asingle, well-defined structure and behavior(Hwang et al. 2009).There are also several limitations to considerwith smFRET. First, and perhaps most fundamentally, this technique generally reports on only asingle dimension of interest, though strides havebeen made toward extending it to two or moredimensions (see “Recent Developments andExtensions of smFRET,” below). Due to thisfact, data from smFRET experiments must becarefully interpreted in light of all available structural and functional information about the system,such as that obtained by X-ray crystallography,mutational studies, and structural footprinting.Second, the millisecond time resolution is tooslow to detect some important molecular dynamics. Third, only 105 photons can be collectedfrom even very stable organic fluorophores beforephotobleaching occurs so that a finite number ofobservations can be made on a given molecule.Furthermore, even with the addition of antiblinking agents such as Trolox, the acceptor may7occasionally enter a dark state, giving the appearance of an excursion to a state with Eapp 0; suchdark states should be excluded from analysis ortheir influence quantified using controls (Benítezet al. 2010).Another concern is that rotational constraint ofthe donor or acceptor fluorophore can interferewith accurate distance measurements; forinstance, common organic fluorophores havebeen shown to stack at the ends of nucleic acidduplexes, influencing the apparent FRET efficiency. To reduce the likelihood of such interactions, a short flexible organic linker (generally analkyl moiety) can usually be added between themolecule of interest and each fluorophore. Still, ifaccurate absolute distance information is required,fluorescence anisotropy measurements are neededto ensure that the anisotropy values of the donorand acceptor are acceptably low (generally 0.2).If only relative distance information is needed,higher anisotropy values are tolerable since Eappis still generally a monotonic function of distance(Roy et al. 2008). Finally, it is important to verifythat the surface immobilization, if used, does notperturb the behavior of interest; this can be doneby comparing results with those of ensembleFRET assays in solution, conducting singlemolecule activity assays, or comparing resultsusing different immobilization strategies.Recent Developments and Extensions ofsmFRETAs the complexity and heterogeneity of moleculardynamics becomes more apparent, efforts are inprogress to extend the capabilities of smFRET andcombine it with other techniques for a more comprehensive real-time picture of molecular events.For instance, three-color smFRET has been developed to simultaneously monitor the distancesbetween a single donor and two distinct acceptorfluorophores (Roy et al. 2008). A challenge inextending such approaches to measure a largernumber of distances is finding multiple acceptorswhose emission spectra are sufficiently distinct toresolve their signals. To partially bypass thisissue, switchable smFRET was developed to
8monitor energy transfer from one donor to multiple acceptors by photochemically switching eachacceptor on and off in succession (Uphoffet al. 2010).Recently, smFRET has also been used in conjunction with other single-molecule techniques, asproposed by Shimon Weiss (Weiss 1999). Forexample, a combination of smFRET and electricalrecording was used to monitor dimerization ofsingle ion channels, thus simultaneously providing structural and functional information. Opticalor magnetic trapping has been used to manipulatesingle molecules while monitoring their dynamicsby smFRET (Hwang et al. 2009).Finally, efforts are in progress to performsmFRET measurements in vivo to study thebehavior of molecules in their native environment. Such efforts are complicated by the significant background autofluorescence within the cell,as well as the limited ability to control the photophysics of fluorophores in vivo, as is done in vitrothrough oxygen scavenging and additives. Nevertheless, smFRET has been employed to study asmall number of intracellular systems, includingprotein–protein interactions at the cell membrane,where TIRF illumination can significantly reducebackground fluorescence. A step in that directioncan also be the immunoprecipitation from a cellextract of a biomolecular assembly of interest inwhich two fluorophores are judiciously placed, atechnique that has been coined single-moleculepulldown FRET or SiMPull-FRET (Krishnanet al. 2013).SummarySingle-molecule FRET is a powerful techniquewith the unique ability to monitor dynamic processes in single molecules over distances of 10 nm and timescales of milliseconds to hours.It exploits the steep distance dependence ( r6) ofFörster energy transfer and high sensitivity ofratiometric fluorescence detection to measure thedistance between specific molecular sites overtime in single molecules. Although it requiressome specialized equipment, smFRET is nowwidely used and has revealed kinetics,Single-Molecule Fluorescence Resonance Energy Transfermechanistic details such as transiently visitedstates, and heterogeneous behaviors that aremasked in traditional assays by ensembleaveraging.Cross-References CNS (Crystallography and NMR System) Fluorescence and FRET in Membranes Fluorescence Labeling of Nucleic Acids Helicases Hidden Markov Modeling in Single-MoleculeBiophysics Magnetic Tweezers Optical Tweezers Patch-Clamp Recording of Single ChannelActivity: Acquisition and Analysis Protein Fluorescent Dye Labeling Single Fluorophore Blinking Single Fluorophores Photobleaching Single-Molecule Methods Single-Molecule Spectroscopy Total Internal Reflection Fluorescence Microscopy for Single-Molecule StudiesReferencesBenítez JJ, Keller AM, Chen P (2010) Nanovesicle trapping for studying weak protein interactions by singlemolecule FRET. Methods Enzymol 472:41–60Blanco M, Walter NG (2010) Analysis of complex singlemolecule FRET time trajectories. Methods Enzymol472:153–178Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR,Weiss PR (1996) Probing the interaction between twosingle molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. ProcNatl Acad Sci U S A 93:6264–6268Hwang LC, Hohlbein J, Holden SJ, Kapanidis AN(2009) Single-molecule FRET: methods and biologicalapplications. In: Hinterdorfer P, van Oijen A (eds)Handbookofsingle-moleculebiophysics,vol 1. Springer, Dordrecht, pp 129–163Krishnan R, Blanco MR, Kahlscheuer ML, Abelson J,Guthrie C, Walter NG (2013) Biased Brownianratcheting leads to pre-mRNA remodeling and captureprior to first-step splicing. Nat Struct Mol Biol20:1450–1457Roy R, Hohng S, Ha T (2008) A practical guide to singlemolecule FRET. Nat Methods 5:507–516
Single-Molecule Fluorescence Resonance Energy TransferSakmann BA, Neher E (eds) (2009) Single channel recording, 2nd edn. Springer, New YorkStryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846Uphoff S, Holden SJ, Le Reste L, Periz J, van de Linde S,Heilemann M, Kapanidis AN (2010) Monitoring9multiple distances within a single molecule usingswitchable FRET. Nat Methods 7:831–836Walter NG, Huang C-Y, Manzo AJ, Sobhy MA (2008) Doit-yourself guide: how to use the modern singlemolecule toolkit. Nat Methods 5:475–489Weiss S (1999) Fluorescence spectroscopy of single biomolecules. Science 283:1676–1683
Single-molecule Förster resonance energy trans-fer; Single-pair fluorescence resonance energy transfer Definition Single-molecule fluorescence resonance energy transfer (smFRET)isatechnique usedtomeasure nanometer-scale distances between specific sites on an individual molecule, usually as a function of time. Introduction
P.R. Selvin (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol.7:730-4. P.R. Selvin (1995) Fluorescence resonance energy transfer. Meth Enzymol246:300-334. J.R. Lakowicz (2006) Principles of Fluorescence Spectroscopy, 3rd edn. Springer. Olympus Resource Center: Fluorescence resonance energy transfer
Specialized Fluorescence Techniques 171 Single Molecule Fluorescence 172 Fluorescence Correlation Spectroscopy 173 Forster Resonance Energy Transfer 173 Imaging and Super-Resolution Imaging (Con-ventional and Lifetime) 174 Instrumentation and Laser Based Fluorescence Techniques 175 Nonlinear Emission Processes in Fluorescence Spectroscopy 176
Introduction 1785 1.1. Aims of This Review 1785 1.2. Questions in Protein Structure and Function 1786 2. Fluorescence Spectroscopy as a Tool for . We then turn to applications of single-molecule fluorescence resonance energy transfer (FRET) to study polypeptide chain collapse in small single-domain proteins under equilibrium
Single-Molecule Fluorescence Resonance Energy Transfer Investigations of Ribosome-Catalyzed Protein Synthesis Daniel D. MacDougall Jingyi Fei Ruben L. Gonzalez, Jr. I. INTRODUCTION Protein synthesis, or translation, is an inherently dynamic process in which the ribosome traverses the open read-ing frame of a messenger RNA (mRNA) template in
Aromaticity: Benzene Resonance Contributors and the Resonance Hybrid The Greater the Number of Relatively Stable Resonance Contributors and the More Nearly Equivalent Their Structures, the Greater the Resonance Energy (a.k.a. delocalization energy) N O O N O O 1/2 1/2 resonance hybrid (true strucutre) resonance contributor resonance contributor
INTRODUCTION Fluorescence resonance energy transfer (FRET) is a process of energy transfer from a fluorescent donor molecule to a fluorescent acceptor without the involvement of a photon (Fo rster, 1948; Stryer, 1978; Van der Meer et al., 1994). One result is that the fluorescence emission of the acceptor
Practical fluorescence microscopy 37 4.1 Bright-field versus fluorescence microscopy 37 4.2 Epi-illumination fluorescence microscopy 37 4.3 Basic equipment and supplies for epi-illumination fluorescence . microscopy. This manual provides basic information on fluorescence microscopy
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