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REVIEW OF SCIENTIFIC INSTRUMENTS VOLUME 74, NUMBER 8 AUGUST 2003

REVIEW ARTICLE

Methods of single-molecule fluorescence spectroscopy and microscopyW. E. Moernera) and David P. FrommDepartment of Chemistry, Stanford University, Mail Code 5080, Stanford, California 94305-5080

~Received 24 September 2002; accepted 21 April 2003!

Optical spectroscopy at the ultimate limit of a single molecule has grown over the past dozen yearsinto a powerful technique for exploring the individual nanoscale behavior of molecules in complexlocal environments. Observing a single molecule removes the usual ensemble average, allowing theexploration of hidden heterogeneity in complex condensed phases as well as direct observation ofdynamical state changes arising from photophysics and photochemistry, without synchronization.This article reviews the experimental techniques of single-molecule fluorescence spectroscopy andmicroscopy with emphasis on studies at room temperature where the same single molecule isstudied for an extended period. Key to successful single-molecule detection is the need to optimizesignal-to-noise ratio, and the physical parameters affecting both signal and noise are described indetail. Four successful microscopic methods including the wide-field techniques of epifluorescenceand total internal reflection, as well as confocal and near-field optical scanning microscopies aredescribed. In order to extract the maximum amount of information from an experiment, a wide arrayof properties of the emission can be recorded, such as polarization, spectrum, degree of energytransfer, and spatial position. Whatever variable is measured, the time dependence of the parametercan yield information about excited state lifetimes, photochemistry, local environmentalfluctuations, enzymatic activity, quantum optics, and many other dynamical effects. Due to thebreadth of applications now appearing, single-molecule spectroscopy and microscopy may beviewed as useful new tools for the study of dynamics in complex systems, especially whereensemble averaging or lack of synchronization may obscure the details of the process understudy. © 2003 American Institute of Physics.@DOI: 10.1063/1.1589587#

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I. INTRODUCTION

Single-molecule spectroscopy~SMS! allows exactly onemolecule hidden deep within a condensed phase sampleobserved by using tunable optical radiation@Fig. 1~a!#. Thisrepresents detection and spectroscopy at the ultimate stivity level of ;1.66310224 moles of the molecule of interest ~1.66 yoctomole!, or a quantity of moles equal to thinverse of Avogadro’s number~a ‘‘guacamole’’!.1 To probethe molecule, a light beam~typically a laser! is used to pumpan electronic transition of the one molecule resonant withoptical wavelength@Fig. 1~a!#, and the resulting optical absorption is detected either directly2 or indirectly by fluores-cence excitation.3 Detection of the single molecule of interemust be done in the presence of billions to trillions of solveor host molecules and in the presence of noise from the msurement itself. The field of SMS has grown over the pdozen years to the status of a powerful technique for exping the individual nanoscale behavior of molecules in coplex local environments.4,5

How can SMS provide new information? Clearly, stadard ensemble measurements which yield the average v

a!Author to whom correspondence should be addressed; [email protected]

3590034-6748/2003/74(8)/3597/23/$20.00

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of a parameter for a large number of~presumably identical!copies of the molecule of interest still have great value.contrast, SMS completely removes the ensemble averagwhich allows construction of a frequency histogram of tactual distribution of values~i.e., the probability distributionfunction! for an experimental parameter. It is clear that tdistribution contains more information than the averavalue alone. For example, the shape of the full distribut~indeed all the moments of the distribution! can be examinedto see if it has multiple peaks, or whether it has a stronskewed shape. Such details of the underlying distributbecome crucially important when the system under studheterogeneous. This would be expected to be the casemany complex condensed phase environments such ascrystals, polymers, or glasses. Fortunately, a single molecan be a local reporter of its ‘‘nanoenvironment,’’ that is,the exact constellation of functional groups, atoms, ioelectrostatic charges and/or other sources of local fields inimmediate vicinity. For biomolecules, heterogeneity easarises, for example, if the various individual copies of a ptein or oligonucleotide are in different folded states, differeconfigurations, or different stages of an enzymatic cycle.

Another advantage of SMS measurements is that tremove the need for synchronization of many single mecules undergoing a time-dependent process. For exampil:

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3598 Rev. Sci. Instrum., Vol. 74, No. 8, August 2003 W. E. Moerner and D. P. Fromm

FIG. 1. ~Color! ~a! Schematic of an optical beam focal region~triangles! pumping a single resonant molecule, which subsequently either removes phfrom the pumping beam or emits fluorescence.~b! Typical energy level scheme for single-molecule spectroscopy.S0 , ground singlet state;S1 , first excitedsinglet;T1 , lowest triplet state or other intermediate state. For each electronic state, several levels in the vibrational progression are shown. Pumpatenergy hn excite the dipole-allowed singlet–singlet transition. The intersystem crossing or intermediate production rate iskISC , and the triplet decay rate iskT . Fluorescence emission shown as dotted lines originates fromS1 and terminates on various vibrationally excited levels ofS0 or S0 itself.

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large ensemble of molecules undergoing intersystem cring events must be synchronized in order to measuretriplet lifetime, and this synchronization is only completethe initial instant. Similarly, an enzymatic system may beone of several catalytic states, and in an ensemble meament initial synchronization is required but is quickly lostthe subsequent dynamical transitions of the individualzymes are stochastic and generally uncorrelated. Howevsingle copies are observed, any one member of the enseis in only one state at a given time, and thus the specsequence of state changes arising from binding, hydrolyand other catalytic steps is available for study. With protime resolution, rare intermediates can be directly probwhereas in the ensemble regime these fleeting species alow concentration and can be swamped by other, more polated species. Using polarized excitation and/or polarizaanalysis of emission, the orientation of a molecular transitdipole can be used to follow mechanical changes such asmotion of biomolecular motors during the force generatprocess.

A final reason for the use of single-molecule techniquis the possibility of observing new effects in unexploredgimes. For example, several single-molecule systems hunexpectedly shown some form of fluctuating, flickering,stochastic behavior.6 The absorption frequency of the singmolecule can change as a result a change in its photophyparameters or a change in local environment; this behahas been termed ‘‘spectral diffusion,’’ and it can produspectral shifts or fluctuations. Such fluctuations are nowcoming important diagnostics of the single-molecule regimand they provide unprecedented insight into behavior whis generally obscured by ensemble averaging. On a funmental level, the time-dependent dynamical behavior ofindividual quantum system would be expected to sh‘‘quantum jumps’’7 as transitions between states occur, athis can be directly observed by SMS techniques.8

Stepping back for a moment, the last few decades h


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witnessed a dramatic increase in interest in the ‘‘nanoworof single atoms, ions, and molecules, for both scientific atechnological reasons. Indeed, SMS as defined is relatebut distinct from, several recently successful lines ofsearch on individual species:~i! the spectroscopy of singleelectrons or ions confined in electromagnetic traps;9–11 ~ii !scanning tunneling microscopy,12 and atomic forcemicroscopy13 of atoms and molecules on surfaces;~iii ! thestudy of ion currents in single transmembrane channel14

~iv! single polymer or DNA chains with high concentrationof fluorophores;15 and~v! force measurements on single mlecular motors using optical traps.16,17

It is useful to note that at room temperature and unappropriate dilution conditions one can detect the burstemitted photons as a single fluorescent molecule in flowsolution passes through the focus of a laser beam.18–21 Thisarea was pioneered by the Keller group at Los Alamos,has been the subject of a recent monograph.22 A related tech-nique, fluorescence correlation spectroscopy~FCS!, usesBrownian motion to bring single molecules through a fcused laser spot. By calculating the autocorrelation ofemitted bursts of light, time-dependent dynamics can belowed in detail.23–29However, since the emission bursts fromany single molecules are summed in the autocorrelatdifferences between individuals may be hard to detect. Inarticle, the focus is on microscopic imaging methods, whone observes the same single molecule for an extendedriod ~longer than the diffusion time through the laser focu!,measuring signal strength, lifetime, polarization, fluctuatioand so on, all as a function of time. Nevertheless, the anses of signal, noise, background, and detection consiations presented in Sec. II below apply directly to detectproblems in flowing streams and FCS as well.

At present, the impact of SMS spans several fields, frphysics, to chemistry, to biology, and the number of applitions continues to expand. In this article, the primary expemental techniques for single-molecule fluorescence detec


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3599Rev. Sci. Instrum., Vol. 74, No. 8, August 2003 Molecular spectroscopy and microscopy

and microscopy at room temperature will be reviewed. Afa summary of the basic principles controlling the signal-noise ratio, a variety of microscopic configurations will bdescribed. This will be followed by a short descriptionmany of the detection modalities that have been developeorder to extract information from a single-molecule opticexperiment. Several comprehensive reviews of this areabe consulted for details of the scientific results that habeen obtained by researchers around the world.4,5,21,22,30–40

II. BASIC PRINCIPLES

How is it possible to use optical radiation to isolate aprobe a single impurity molecule on a surface, in a liquid,inside a solid sample? To answer this question concissingle-molecule optical spectroscopy is accomplished bydeceptively simple steps:~a! guaranteeing that only one moecule is in resonance in the volume probed by the laser,~b! providing a signal-to-noise ratio~SNR! for the single-molecule signal that is greater than unity for a reasonaaveraging time. These two requirements will now be treabriefly, and then in more detail.

At room temperature, guaranteeing that only one mecule is in resonance is generally achieved by a combinaof focusing the laser to a small probe volume(;1 – 100mm3, even down to 1024 mm3 for near-fieldmethods!, using an ultrapure host matrix, and selectingultralow concentration of the impurity molecule of intereThe concentration required depends upon the volume proby the pumping laser, which varies somewhat dependingthe technique and the sample under study as describemore detail below. For example, at room temperatureneed only work with roughly 10210 mole/L concentration ina probed volume of 10mm3 to achieve the limit of one mol-ecule in resonance. At liquid helium temperatures, the pnomenon of inhomogeneous broadening41–43 can be used toachieve dilution factors from;104– 105 simply by tuningthe laser frequency to a spectral region where only one mecule is in resonance.30,38 This spectral selection techniquwas crucial to the first demonstrations of SMS.2,3 As thisarticle concentrates on room temperature methods, thetailed considerations33,44,45 that apply to low temperaturespectral selection techniques will not be described furthecartoon of the selection process is shown in Fig. 1~a!, wherea focused laser beam selects only one molecule and SMachieved by detection of the photons from this molecule

Achieving the required SNR requires close attentionmaximizing signal while minimizing backgrounds and trelative size of laser shot noise. To obtain as large a signapossible, one needs a combination of small focal volumlarge absorption cross section, high photostability, webottlenecks into dark states such as triplet states, and option below saturation of the molecular absorption. For flurescence methods, one must select an impurity moleculethe highest fluorescence quantum efficiency as possibleaddition, one must rigorously exclude undesirable fluorcent impurities, minimize the volume probed to reduce Rman scattering, and scrupulously reject any Rayleigh stered radiation at the pumping wavelength. For absorp


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methods, achieving a low noise level from nonideal effefollows from careful reduction of residual signals and opetion at a power level sufficient to reduce the relative conbution from laser shot noise, see Refs. 30, 44, and 46details.

A. Signal size

The signal of interest here arises from the fluorescphotons emitted by the single molecule. While the numbephotons emitted per second for one molecule is far smathan the number of photons per second in the incident labeam, the fact that the emitted photons are generallyshifted to longer wavelengths enables detection with reasable SNR. Figure 1~b! shows the salient features of the mocommon energy level structure of molecules that have bexplored by SMS. The molecule has an electric–dipoallowed singlet–singlet optical transitionS1←S0 , pumpedby radiation at energy hn from a laser or a lamp, with h thePlanck constant andn the optical frequency. It is generallnot necessary to pump the lowest electronic excited stateabsorption into a vibrationally excited sideband is sufficieAfter the absorption of the pump photon, the excited mecule quickly relaxes by emission of vibrational modes ofmolecule~intramolecular vibrational relaxation! and of thehost ~phonons! to the lowest electronic excited state, frowhich fluorescence photons can be emitted~dashed lines!.~There is also a certain probability for intersystem crossinto the triplet state manifold temporarily,vide infra.! Afterthe emission, the molecule is brought back to the initground state by further vibrational and phonon relaxatiand the partitioning between the various colors of emissiocontrolled by the well-known Franck–Condon and DebyWaller factors expressing the extent of vibrational relaxatand electron–phonon coupling, respectively. In general,relaxation steps represent energy losses which cause atral redshift between absorption and emission bands, cathe Stokes shift. Typical fluorescent molecules used for Scome from the general classes of laser dyes used in biolcal fluorescent labeling~rhodamines, cyanines, oxazineetc.!,47 rigid aromatic hydrocarbons such as terrylene,48 orperylene diimides,49,50as well as autofluorescent, geneticaexpressed proteins such as the green fluorescent pr~GFP! and its relatives.51,52Some proteins are naturally fluorescent due to presence of one or many fluorophores asfactors, and these have also provided useful single-molesignals in the cases of flavoenzymes53,54 and antenna complexes containing bacteriochlorophyll molecules.55 In recentwork, a new class of single-molecule fluorophores has bfound among molecules originally optimized for nonlineoptical properties.56 The degree of redshift of the emissiovaries, but the laser dyes for example often undergo larearrangements of their charge distribution upon excitatileading to much larger Stokes shifts~about 1500 cm21 forrhodamine dyes, 6000 cm21 for DCM, another well-knownlaser dye! than for aromatic hydrocarbons. Strong fluorecence has also been observed for single semiconductor qtum dots,57 and these emitters have been proposed as biolcal labels.58 This approach requires methods for attachmof the dots to biomolecules, an area of intense current


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search. From the photophysical point of view, these fasciing emitters behave like two-level systems on short tiscales,59 have narrow emission lines and resist photobleaing, yet show fluorescence intermittency and blinking ovmany decades in time.60,61

To maximize the fluorescence emission rate, it is necsary to pump the molecule with high probability and to hathe largest possible fluorescence quantum yieldfF . For fo-cal spots of area greater than or equal to the diffracti

limited value ~approximately (l/2)2, with l the opticalwavelength!, the rate at which the resonant optical transitiis pumped is given by the product of the incident photon fl@in photons/(s cm2)# and the absorption cross sectionsp ~incm2). Stated differently, the probability that a single moecule will absorb an incident photon from the pumping labeam is justsp /A, whereA is the cross-sectional area of thfocused laser beam. Thus,sp may be interpreted as the efective ‘‘area’’ ~per molecule! which is able to ‘‘capture’’photons from the incident laser beam. Highsp means thatthe photons of the incident light beam are efficiently asorbed and therefore background signals from unabsophotons are minimized compared to the case for weaksorbers. Thus it comes as no surprise that maximizingabsorption cross section is crucial to the detection of sinmolecules.

Generally, the maximum values ofsp arise fromstrongly allowed electric dipole transitions, and the valuethis quantity is intimately connected to the quantumechanical details of the electronic transition, specificallythe transition dipole moment~or equivalently, the radiativerate given by the EinsteinA coefficient!. The general expression for the ~peak! cross section for a randomly orientemolecule is

sp52p~l/2p!2~g r /G tot!, ~1!

wherel is the light wavelength,g r the spontaneous~that is,radiative! fluorescence rate, andG tot the total frequencywidth of the absorption.4,44,62Happily, most electric–dipole-allowed transitions in organic molecules have oscillastrengths on the order of unity. For a dye molecule in sotion at room temperature withl5500 nm, g r is about1023 cm21, which corresponds to a radiative lifetime offew ns, andG tot is about 1000 cm21, so that the absorptioncross section of a single dye molecule issp;4 Å254310216 cm2, a value comparable to the molecular size.somewhat more convenient way to estimate the value ofabsorption cross section at room temperature is to usewell-known connection between molecular extinction coecient« ~units L mol21 cm21, tabulated and easily measure!and the cross section63

s52.303«/NA ~2!

in units cm2, with NA the Avogadro constant.@At low tem-peratures, optical absorption profiles become extremelyrow so that all molecules in the sample may not be resonwith the pumping laser, and more careful methods musused to determine the cross section.44,64 In this case, the tota


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width of the absorption becomes so narrow that cross stions often approach the fundamental maximum valuel2/(2p).] 38

The other key molecular parameter controlling tamount of emitted fluorescence is the probability for phoemission per absorption event, i.e., the fluorescence quanyield fF . This parameter should clearly be high, as closeunity as possible. The fluorescence quantum yield is geally given by

fF5krad/~krad1knonrad!5tF /t rad, ~3!

wherekrad is the radiative rate~EinsteinA coefficient!, knonrad

is the sum of all nonradiative rates such as internal convsion or intersystem crossing,tF is the observed excited statlifetime ~fluorescence lifetime!, and t rad is the radiativelifetime.63 Thus, the best fluorophores are those with retively rigid structures so that the primary path for deactivtion of the excited state is via the emission of a fluorescphoton. Molecules that twist or distort greatly in the excitstate typically have non-negligible nonradiative decay chnels. For a strong emitter~and absorber! like R6G wherefF;0.45 ~in water!,65 the fluorescence lifetime is;4 ns.This means that if the molecule were a two-level system,maximum emission rate would be on the order of 108 s21.Although this is quite a large signal, several effects actlimit the maximum photon emission rate and the maximunumber of photons emitted, which will now be described

The first physical effect that limits the maximum emision rate from a molecule is optical saturation of the trantion. As laser power is increased, more and more photonsemitted per second, as long as the optical transition issaturated. When saturation occurs, the absorption crosstion from the molecule decreases, and further increaselaser power generate more background photons rathersignal photons. More precisely, the cross section for abstion depends upon pumping laser intensityI as

sP5sP0 /~11I /I S!, ~4!

whereI S is the characteristic saturation intensity. The satution intensity depends upon further details of the energy lestructure of the molecule. In the limit that the molecule aproximates a two-level system, the saturation intensitygiven by

I S5hn/~2stF!. ~5!

This equation expresses the fact that if the rate of absorpof incident photons gets too high, the molecule cannot deback to the ground state fast enough, thus the ability ofmolecule to absorb photons decreases.

Although some fluorophores do approach the two-lelimit, it is generally more appropriate to consider the threlevel case shown in Fig. 1~b!. The intersystem crossing process shown may be regarded as a model for any bottlenthat takes the molecule into a form that no longer absorbemits photons. In organic molecules, intersystem cross~ISC! is seldom rigorously absent, and when ISC occuboth absorption of photons and photon emission cease frelatively long time equal to the triplet lifetime (51/kT).This effect results in premature saturation of the emiss


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rate from the molecule and reduction of the absorption crsectionsp compared to the two-level case.66 The saturationintensity for a molecule with a triplet bottleneck may bwritten45,67

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wherek2151/t21 is the rate of direct decay fromS1 to S0 ,kISC is the rate of intersystem crossing, andkT is the totaldecay rate from the lowest tripletT1 back toS0 . Clearly, thismore accurate saturation intensity is smaller than the tlevel value by the bracketed factor on the right, and smasaturation intensities mean the laser power that can beto probe the system is limited~for fixed focal spot area!.

The effect of saturation can be seen in both the pon-resonance emission rate from the moleculeR(I ) and inthe single-molecule linewidthDn(I ) by solving the three-level rate equations with the result36,66

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R`5~k211kISC!fF

21~kISC/kT!. ~9!

Equations~7! and~8! show that the integrated area undthe single-molecule peak falls in the strong saturationgime. At the same time, at higher and higher laser powmore and more scattering signal is produced in~linear! pro-portion to the laser power, so the difficulty of detectingsingle molecule increases. This specific effect will be eplored more directly in the SNR discussion in Sec. III C. Tdependences of the maximum emission rate and linewidthlaser intensity in Eqs.~7! and ~8! have been verified experimentally for individual single molecules.66 In summary,minimizing the triplet bottleneck means small values ofkISC

and large values ofkT , requirements which are satisfied forigid, planar aromatic laser dye molecules, aromatic hydcarbons, and other good fluorophores. In cases wherephotophysical parameters of all bottleneck states areknown, it may be more convenient to directly measuresaturation intensity by recording and fitting the intensidependent transmission of a bulk sample to determineI S .68

In particular, for real molecules at room temperature, sittions may occur where rapid relaxation and decoherencthe vibrational manifolds should be taken into account, anmay become necessary to go beyond the three-level mpresented here.

A final physical effect that limits the~integrated! signalfrom a single molecule is photobleaching, a general termany photochemical process that causes the molecule to etually change to another form and stop absorbing andemitting photons. In practice, at room temperature almosmolecules in aqueous solution photobleach after the emisof ;106 photons. Specific values of the photobleachiquantum yieldfB are provided, e.g., in Refs. 65 and 6generally the maximum number of photons emitted on av


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age is given by 1/fB . In many cases, photobleaching is atually a photo-oxidation of the fluorophore, so measuresreduce the presence of molecular oxygen can help in mmizing this effect. Successful methods have involved:~a! theuse of an enzymatic oxygen scavenger system,70,71 ~b! flood-ing the sample with an inert gas such as argon or nitroge72

and ~c! operating in vacuum.73

B. Background issues

To detect a single molecule, one must simultaneoumaximize signal while at the same time scrupulously mimize background photons, noise from unwanted sources,detector dark counts. For the purposes of this discuss‘‘noise’’ refers to fluctuations arising from effects suchPoisson statistics of detected photons, while ‘‘backgrounrefers to photons that may arrive at the detector from asource other than the single molecule of interest. Backgrosignals generally scale linearly with the pumping laspower, while detector dark counts do not.

In spite of much effort to reduce backgrounds, almostsingle-molecule fluorescence experiments are backgrolimited and the shot noise of the probing laser is only imptant for the signal-to-noise of the spectral feature. Therefoconquering background signals must be one of the primgoals of the single-molecule experimental design. A fisource of background may result from experimental limitions such as the residual fluorescence from optical paparticularly from colored glass filters or from the microscoobjective itself. It is also possible to have residual emissfrom the excitation source in the red-shifted spectral ranwhere fluorescence is detected. This source of backgrocan be a critical issue when the laser in use is a diode laand multiple filters may be required to suppress laser emsion to long wavelengths of the primary mode. In general,careful selection of high-quality components, these sourof background may be minimized.

However, background photons arising from the samitself are more difficult to suppress. These are of two typelastic Rayleigh scattering of the laser wavelength, and ptons that are red-shifted in wavelength into the spectralgion of the single-molecule fluorescence and hence thatobscure the photons from the single molecule. Rayleiscattered pump radiation can be reduced by using ultraclscratch-free substrates of the highest quality and by theof interference filters as described below. The two msources of redshifted photons are residual fluorescenceother impurities, and Raman scattering by the solvent or hmatrix. Both are proportional to the volume of the illumnated sample. Even if their fluorescence yield is very weunwanted impurities or molecular components of the hmedium, particularly in biological samples, can emit stroresidual fluorescence due simply to the fact that billionstrillions of host molecules may be present in the illuminatvolume. Thus it is necessary to use ultrapure solventsnanopure water for sample preparation. Residual fluocence increases very rapidly with the energy of the excitphoton, and thus working in the blue or near-ultraviolet~UV!part of the spectrum will often give rise to more seriobackground problems. To illustrate the issues with Ram


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scattering, one may consider benzene as a solvent. TheRaman scattering cross-section of benzene for one ostrongest modes is about 10212 Å 2 ~per molecule!,33 whichgives a total cross section of less than 1022 Å 2 for an ex-cited volume of 1mm3. This shows that Raman scatterincan become comparable to the absorption cross sectionsingle molecule in a solution at room temperature for excivolumes of about 100mm3. Given that the background crossections are proportional to the excited volume, an optimsignal-to-background ratio will require a minimum excitevolume, and all of the microscope configurations describin Sec. III work toward this goal.

As mentioned above, filters are generally required inder to suppress Rayleigh scattering of the pump radiationillustrate, suppose a single molecule of rhodamine 6Gprobed with 1 kW/cm2, somewhat below the onset of satration of the absorption. The resulting incident photon flux532 nm of ;331021 photons/s cm2 will produce about 13106 excitations/s. With a fluorescence quantum yield0.9, about 93105 emitted photons/s can be expected. At tsame time,;331013 pump photons/s illuminate a focal sp1 mm in diameter. It is of course helpful in reducing the siof Rayleigh scattered pump radiation at the detector that ptons are often collected in the backward direction. In acase, rejection of the Rayleigh scattered pumping radiaby a factor on the order of 107– 108 is generally required,with minimal attenuation of the fluorescence.

Thus, filtering for single-molecule experiments musttenuate Rayleigh scattering by the largest possible fawhile transmitting the desired redshifted fluorescence frthe single molecule. If additional suppression of host/maRaman scattering is desired, the transmission window offilters can be further optimized to reject any strong~Stokes-shifted! Raman lines, but this must be done in a way thdoes not attenuate the precious fluorescence photonsthe single molecule unduly. The required filtering for a spcific experiment is generally accomplished by~in increasingorder of performance! low-fluorescence glass filters~Schott!,interference filters~Chroma, Omega! or by relatively expen-sive holographic notch attenuation filters~Kaiser!. The filtercharacteristics must have a high slope in the filter transmsion spectrum in order to open quickly enough as wavelenis increased to pass the single-molecule emission. Figu

FIG. 2. Example of filtering considerations for SMS. The left panel shotransmission spectra of a 532 nm holographic Raman notch filter, a 550long pass emission filter, and the transmission of the dichroic beamspthrough which the fluorescence must pass. The right pane shows thesion spectrum of Nile red in methanol solution, and the resulting shapthe emission spectrum after passage through all three filters. The integarea ratio shows that 46% of the fluorescence collected by the microsobjective passes through the filters.


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illustrates the filter characteristics for a set of filters chosfor microscope experiments with the dye nile red pumped532 nm. The left side of the figure shows transmission sptra of: ~a! a 532 nm holographic Raman notch filter,~b! a 550nm long pass emission filter, and~c! a 540 nm dichroicbeamsplitter through which the emitted fluorescence mpass~see microscope descriptions in Sec. III!. The product ofthese three transmission spectra gives the wavelength dedent transmission factorFfilter that must be applied to thesingle-molecule fluorescence spectrum. Figure 2~right side!shows the unperturbed nile red emission spectrum~uppertrace!, and the spectrum after passage through the threeters~lower trace!. By comparing the areas of the two curveFfilter546% of the nile red emission is passed on to thetector. The transmission at the pump wavelength is so smthat it cannot be easily measured with a conventional UV-spectrometer, so direct measurement of pump laser transsion is preferrable.

C. SNR and signal to background ratio „SBR…

While many applications of fluorescence imaging haneed of high signal-to-noise ratios, few are as demandinsingle-molecule imaging at ambient temperatures. Now tthe fundamentals of single molecule emission and the baground issues have been described, the full set of factorsaffect the SNR for single-molecule fluorescence experimemay be presented in a more quantitative fashion. Figure~a!illustrates the basic definitions of the various signal abackground levels. In all experiments, there is a control vable ~abscissa! that has the effect of making the singlemolecule signal appear and disappear. For example, thetrol variable in a microscopic experiment would be spatposition defined either by the position of the focal volumeby the spatial position on the two-dimensional detector.~Inthe earlier low temperature experiments, the laser freque

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FIG. 3. SNR considerations:~a! for a hypothetical signal with control vari-able shown on the horizontal axis, the definitions of the offset, dark leand background levelB are shown. The signal from the single moleculeS. ~b! Surface plot of fluorescence SNR vs probing laser power and fospot area for a fluorophore with the following parameters:l5532 nm, t50.1 s, fF50.28 ~RG6 in water, Ref. 65!, I S55.63103 W/cm2 ~Ref. 69!,s54310216 cm2, D50.072,Cb523108 counts/W s~measured in a typi-cal confocal microscope!, Nd5100 counts/s.~c! SNR for the same modefluorophore assuming a photobleaching quantum yield of 1025.


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or the local applied electric field could also act as the convariable.! Starting from the zero level, some detectors havconstant dc offset value that is present even if there weredark counts in the presence of no light. This offset is comon in charge coupled device~CCD! detectors, and arisefrom the input offset of the sample and hold amplifier befothe analog-to-digital converter. For all quantitative calcutions, levels should be measured relative to this offset leAbove this offset level is the signal corresponding to the dcounts of the detector. The number of dark counts recorshould increase linearly with the counting timeT, hence theproper value for the average dark count rate can be demined by recording without light for a range ofT values.The background levelB appears on top of the dark coulevel as shown in Fig. 3~a!. If the source of backgroundvaries from spot to spot, then the averageB level will not bethe same at all values of the control variable. Finally,single-molecule signalS appears on top of the sum of thbackground and dark levels.

The fundamental detection problem reduces to thisorder to observe the single-molecule signalS, the size ofSmust be compared to the fluctuations inS, B, and dark,because it is only when the fluctuations are large thatsingle-molecule signal is obscured.~That is, if theB andNlevels were perfectly fixed, then they also would act likesimple offset that can be trivially subtracted from the dat!To express this quantitatively, Basche´ et al. have presentedthe following equation to compute the SNR for fluorescendetection of a single molecule,74 based on the assumptiothat the noise factors limiting detection are the Poisson~shot!noise fluctuations of the single-molecule signal, the baground signal, and the dark counts:

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where fF is the fluorescence quantum yield of the fluorphore, sp is the absorption cross section,T the detectorcounting interval,A the beam area,P0 /hn the number ofincident photons per second,Cb the background count ratper watt of excitation power, andNd the dark count rate.D isan instrument-dependent collection factor which is a prodof the angular collection factorFcoll of the detection system@primarily dependent on the numerical aperture~NA!#, theoptical losses in optics expressed as a transmission faFopt, the filter transmissionFfilter ~see the previous section!,and the detector quantum efficiencyhQ ; D5hQ Fcoll Fopt

Ffilter . For a detailed discussion of theFcoll factor for a singlemolecule taking into account the dipole radiation pattetotal internal reflection, and the molecular orientation, sRef. 45. This factor depends upon the orientation of the mlecular transition dipole moment relative to the propagatdirection of the incident light. If precise detail of the colletion is required, it is necessary to take into account the mofications of the dipole emission pattern that arise as a reof passage through dielectric interfaces of varying indices


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refraction; molecular emission is more efficient into higindex media.75 Typical values ofD are in the range of1%–8% for modern systems.

Assuming the collection efficiencyD is maximized, Eq.~10! shows that there are several physical parametersmust be chosen carefully in order to maximize the SNFirst, as stated above, the values offF andsp should be aslarge as possible, and the laser spot should be as smapossible. Higher powers produce higher SNR values, butpower P0 cannot be increased arbitrarily because saturacauses the peak absorption cross section to drop fromlow-power value as in Eq.~4!. Various limits of Eq.~10! maybe examined; for example, supposing that backgrou@dark counts, then the SNR at low power grows with tsquare root of power, peaks, and then eventually fallswhen the saturated single-molecule signal becomes smthan the background signal.

To illustrate graphically some of the tradeoffs inherentEq. ~10!, parameters for a model laser dye in the rhodamfamily will be used to explore the SNR function in mordetail. Assuming 0.1 s integration time and using a measuvalue of the background parameterCb in a typical confocalexperiment (;23108 photons/s W), Fig. 3~b! shows thecomputed SNR versus laser power and beam area. It is cthat for a fixed laser spot size, an optimal power exists whmaximizes the tradeoff between the saturating fluorescesignal and the linearly~with P0) increasing background signal. Following the surface plot at a fixed value ofA, the SNRat first improves with increasing power, because the sigincreases linearly withP0 and the shot noise from the powedependent terms in the denominator only grow as the sqroot of the laser power. As saturation sets in, however,SNR falls because the signal no longer increases. Anorelationship illustrated in Fig. 3~b! is that the best-case SNRat smaller and smaller beam areas levels off~the flattening ofthe ridge at small beam area!. This is due to the effect ofsaturation and shot noise—at smaller and smaller areaspower must be reduced eventually to the point whereSNR is controlled by the shot noise of the detected sig@first term in the denominator of Eq.~10!#.

It is important to emphasize that Fig. 3~b! illustrates thebest case, in which the SNR is limited only by the Poissfluctuations inherent in detecting photons. In practice, thare other effects that come into play which may act to redthe attainable SNR in timeT below the optimal values at thpeak of the mountain in the figure. The most important liiting effect usually turns out to be photobleaching. Consida given point on the surface plot defined by a laser powand a spot area, i.e., a particular value of laser intensityintensity is too high, then the molecule may not last loenough to provide photons throughout the counting interT. Typical values of the photobleaching quantum yieldfB

are in the range 1025– 1027,65,69 and the inverse of this parameter effectively determines the total number of emitphotons on average before photobleaching. To approximthe effect of photobleaching, Fig. 3~c! is a calculation iden-tical to Fig. 3~b!, except that when the number of emittephotons exceeds 105 (fB set at 1025 for simplicity!, theSNR is set to zero. The effect on the achievable SNR


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Faced with limitations due to photobleaching, the expementer should make the power/time/SNR tradeoff carefuBy controlling the laser intensity, the rate at which photoare emitted can be easily varied up to the maximum rate.experimenter can then choose which time scale is of the minterest: for fast time information, high laser intensitishould be used to give the best possible SNR in short coing intervalsT before photobleaching. If long-time information is needed, it is better to work with much lower intenties and lower emission rates. It is even useful in soexperiments to pulse the probing laser at low duty cycleobserve the molecule only briefly with long intervening daperiods. This is a very useful strategy especially if the ptobleaching process is nonlinear. In all cases, it is besminimize both the dark counts and the background sourceobtain the maximum information about the molecule.

It is worth noting that Eq.~10! applies to both wide-fieldas well as scanning confocal detection systems. In the cawide-field experiments, it is the diffraction-limited focal arethat controls theA parameter and the power through this arwhich gives theP value for the purpose of determining SNRIn the near-field case, on the other hand, the local elecfields around the tip are modified compared to the casefocused Gaussian beams.76 Moreover, the molecular dipoleinteracts with the local electric field directly via the dot prouct squared, and a more detailed analysis is required wtakes into account the field distribution as well as any otquenching interaction between the tip and the molecule.

Another often-quoted measure of signal size is the SBThis parameter, though useful, is not as fundamental asSNR given above. For this reason, definitions of SBR vaone definition~for negligible dark counts! is SBR5~signalof interest1background scattering!/~background scattering!,and another is SBR5~signal of interest!/~backgroundscattering!. The SBR and SNR both depend upon a numof parameters of the system, including incident intensity, clection and detection efficiencies, quality of sample, etc.,the SNR in addition depends critically upon the detectbandwidth determined by the integration timeT. The SBR isindependent of averaging time and is really only a measof the brightness of the molecule compared to the ovequality of the sample and the ability of the detection systto reduce background. On the other hand, the SNR in adamental way senses the ability to detect the single molecompared to the noise fluctuations that are present wmay masquerade as a single molecule. If the backgroundsignal are both large and emission saturation is not occurrthe relative effect of Poisson noise is smaller, and it becomeasier to detect the presence of a single molecule in spitlarge background. Typical values of SBR for experimewith good fluorophores are on the order of 3–10 or more

D. Detectors for single-molecule experiments

As is evident from the above SNR discussion, detectSM fluorescence requires a device that can detect singleton arrivals with minimal dark noise, and the ‘‘photon bu


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get’’ imposed by irreversible photobleaching effects demathat the quantum efficiency be as high as possible for sinmolecule fluorescence detection. There are essentiallyclasses of detectors for SMS experiments: single-elementectors and two-dimensional array detectors. The capabiland characteristics of various examples of each class wildiscussed, as proper detector selection is central to thecess of SMS experiments.

1. Single-element detectors

Early experiments observing the flow of single dye mecules through a tightened laser focus detected photonsa high-quality microchannel plate photomultiplier tub~PMT!,77 since the PMT offered adequate temporal resotion to observe the short bursts of fluorescence as smallmolecules diffused through the laser focus. While PMTsfer suitable temporal response and can have quite low dnoise levels when cooled, they suffer from low quantumficiencies for visible light (,20%), limiting their use inSMS experiments. Further, PMTs require several pieceslow-noise electronics to efficiently detect single photons, aeven with cooling, dark count rates in the 10–100 cps raare common. PMTs do have a large area of detection onorder of 1 cm31 cm, so this may be an advantage in somcases.

As an alternative, recent advances in semiconductor ptodetectors have allowed for the commercial availabilityavalanche photodiodes~APDs! capable of detecting singlephotons in an integrated package that contains on-boardplifiers and cooling, thus diminishing dark counts to levelslow as 25 dark counts/s. These units, called single phoavalanche photodiodes~SPADs! have much higher quantumefficiencies,.60% across the visible spectrum, even aproaching 95% in the near-infrared~IR! as a result of theband structure of Si. The engineering of these detecmakes the SPAD much easier to use than a PMT; the morequires low operating voltages~5 V!, generating the highvoltage for biasing of the APD internally. A detected photis converted to a digital~TTL! output pulse that can be easicounted on a computer-based digital acquisition board. Tycal temporal response for the SPAD is comparable to PMphotons can be detected with an accuracy of;350 ps fullwidth at half maximum~FWHM!; however, the detector haa larger ‘‘dead time’’ between photon arrival events thus liiting the maximum count rate. Faster SPADs are availabut their increased temporal response generally comes aexpense of lower quantum efficiencies. In terms of dacounts, units are commercially available with 100 cps,cps, even down to 25 cps, but the cost increases dramatias the dark count rate goes down. The only disadvantagthe SPAD compared to the PMT for SMS experiments is tthe SPAD detector has a much smaller active area~180 mmdiameter!. This limitation can easily be overcome in SMconfocal or near-field microscopy experiments, since the ssize at the microscope image plane can easily be madunderfill the SPAD detector chip. The small active area heven been used as a confocal aperture in confocal imaapplications. Because of its ease of use and superior quan


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efficiency the SPAD has become the standard single elemdetector for SMS. Further technical details of the SPADdescribed in Ref. 78.

2. Two-dimensional array detectors

For wide-field microscopic applications, any one of seeral ultrasensitive cameras may be used for the detectiosingle-molecule fluorescence; indeed part of the reasonsingle-molecule studies have become widespread is thetinuing advance in CCD detector array technology thatoccurred over the last decade. Even the human eye, wproperly dark adapted, can see the light from a single mecule in the eyepiece of a microscope that efficiently collethe emitted photons. The use of a commercial cameraamateur astronomy has also been used to observe simolecule emission,79 but such detectors are relatively slowread out, sometimes taking minutes to transfer one framecomputer. Here a selection of modern CCD array detectodescribed.

Liquid-nitrogen cooled, back-illuminated Si CCD arradetectors~Princeton Instruments! have been available fosome time for applications in astronomy and spectroscoThe back illumination provides quite high quantum efciency in the red and infrared, up to;70% – 80%. Liquid-nitrogen cooling provides the cold sink for operation atemperature near2120 °C, and this temperature is carefulchosen to enable charge motion and drastically reducecounts, down to levels near 1 electron/pixel/h. Thus, thisother CCD detectors may be viewed as a collection of 23256565,535 high-performance photon counting devicThe major limitations of this type of detector are:~a! the lowtemperature limits the speed at which electrons can beout, and~b! a penalty of 10–20 electrons of read noisepaid for each analog-to-digital conversion. Thus, these detors are best for cases where long averaging times are acable, such as in recording the spectrum of the fluorescefrom a single molecule.80–84 In this situation, the countinginterval is made long enough so that enough photoelectare collected to overcome the readout noise. Naturally,long averaging times, the spikes that arise from cosmic rmust be removed from any images or spectra.

Intensified frame transfer Si CCD cameras, such asI-Pentamax~Princeton Instruments, now Roper Scientifi!have been commercially available since the early- and m90’s. In this design, photons are first detected by a multkali photocathode, and the electron emitted is amplified~‘‘in-tensified’’! in a microchannel plate coupled to thphotocathode by a fiber bundle. The resulting group of etrons hits a phosphor screen to generate photons agafront-illuminated Si CCD detector converts the phosphphotons into electrons, and the electrons from each Cwell are ultimately converted into a digital number usingfast analog-to-digital converter~ADC!. The CCD and/orphotocathode may be Peltier cooled to reduce noise fthermionic emission. The primary advantage of this desigthe multiplication of the detected photoelectron before aread noise penalty is paid. This means that even one detephoton can be recorded, and the readout noise of the Cbecomes less important. As a result, a faster A


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(;5 MHz conversion rate, with a noisier buffer amplifie!can be used, and detectors in this class can run at vframe rates and higher. Continuous readout with very smdead time is enabled by the frame-transfer mode of option, where only half of the CCD is illuminated; afterframe, the illuminated half is shifted into the hidden half, areadout of the hidden half commences during the next imination time. Limitations of this detector arise from thsomewhat lower quantum efficiency of the photocathode mterial, excess noise that may arise from the multiplicatprocess, nonuniform gain over the various channels ofintensifier plate, and the danger of detector damage by exlight.

A sensitive back-illuminated, nonintensified framtransfer CCD camera~Micromax, Roper Scientific! has beenavailable for several years. This design capitalizes onprovements in design of back-illuminated chips so that fasADCs can be used without large increases in readout noIn a typical system, 5 MHz readout rate with 11 electronpixel read noise has been observed. Advantages of thissign are the higher native quantum efficiency of Si, the muhigher degree of uniformity in sensitivity from pixel to pixeand the lack of intensifier-related distortions. Of courwithout multiplication, the camera is better suited fbrighter fluorophores, i.e., the read noise must be overcoto detect photoelectrons.

Very recently, frame-transfer Si CDD cameras with ochip multiplication gain have become available~Cascade,Roper Scientific; iXon, Andor Technology!. This design usesa CCD chip that operates for the most part like a standCCD except that the last row of CCD wells is connected tseries of additional wells on the chip biased so as to havsmall amount of secondary emission as the electronspassed from well to well. The net effect is a multiplicativgain factor of up to;1003 on the chip, before any reapenalty is paid. This detector may find widespread applition since it combines the virtues of Si as a photodetecwith relative insensitivity to readout noise. Moreover, tfully integrated design means that the optical configurationmuch simpler than the intensified CCD in which a photocaode, fiber bundle, microchannel plate, phosphor, and Cmust all be optically cemented together.

Other specialized detectors can be used for singmolecule microscopy. One example is the resistive anmicrochannel plate detector, which is effectively a large-aPMT in which the detected signal is located inx–y positionby a form of spatially dependent readout~e.g., Photek,Roentdek GMBH!. This detector can provide fast timing information, at the expense of lower quantum efficiency anlimited maximum detection rate—each photoelectron mbe swept out of the device before another arrives.

III. MICROSCOPE CONFIGURATIONS

Several optical configurations have been demonstratesatisfy the basic requirements for single-molecule detecand spectroscopy, where the molecule is studied for antended period long compared to the diffusion time througfocused laser spot. Successful microscopic techniques


,

41


FIG. 4. ~Color! Confocal microscopy.~a! Schematic of experimental setup (O) objective, (D) dichroic beamsplitter, (F) filters, (A) confocal aperture torestrict axial extent of the sample that can lead to scattering backgrounds, and~SPAD! silicon photon counting avalanche detector.~b! Example confocal imageof nile red single molecules in a PMMA thin film.l5532 nm, laser intensity 1.1 kW/cm2, 100 by 100 pixel image, 10mm310mm field at the sample plane10 ms dwell per pixel, total acquisition time 100 s. The fluorescence was filtered with a 540DRLP dichroic beamsplitter~Chroma!, E550LP emission filter~Chroma!, Raman Super Notch Plus 532~Kaiser Optical! emission filter, imaged through a 75mm confocal aperture, and detected with a SPCM-AQ-1SPAD ~EG&G!. The point spread function of the microscope is slightly elongated in the vertical direction.

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clude scanning methods such as near-field scanning opmicroscopy ~NSOM! and confocal microscopy and widefield methods such as total internal reflection and epifluocence microscopy.

A. Scanning methods

One family of SMS techniques involves illuminating thsample with the smallest spot possible and scanning ethe illumination spot or the sample itself. An image is costructed point by point, and when an immobilized singmolecule is located, it can be studied by positioning the laspot directly over the molecule. While scanning metholack the ability to observe several isolated single molecuin parallel, and thus prevent real-time observation of motover microscopic distances, the smaller illumination voluused lowers background, improving the SNR. Additionathe detectors used in scanning experiments offer higher tporal resolution than the CCD cameras used in wide-fiexperiments, whose temporal response is limited by the aity to read out the CCD detector array. Thus, for experimethat require maximum sensitivity and temporal resolutioscanning methods are the preferred SMS method. Theprimary scanning methods used will be highlighted beloconfocal microscopy, in which a diffraction-limited lasespot is the point source, and NSOM, where so-called ‘‘nefield’’ effects create a laser spot smaller than the diffractlimit.

1. Confocal microscopy

Figure 4~a! shows a simplified schematic of a confocmicroscope. A collimated laser beam is reflected off odichroic beamsplitter and through an infinity-corrected, hNA microscope objective, with the result that the laserfocused to a diffraction-limited spot at the sample plane. Tdiameter of the laser focus can be defined by several crite


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such as the well-known Rayleigh criterion,85 the Sparrowcriterion, or others. The Sparrow criterion states that tpoints of equal brightness can be distinguished if the intsity at the midway point is equal to that at the points~i.e., thegradient at the peak of the summed profile is zero!, and isgiven by: ds50.51l/NA, where l is the excitation wave-length. Thus, forl5488 nm a NA51.4 ~oil! objective willgive ds5178 nm, while a top-quality air objective, NA50.9 produces a spot withds5277 nm. Since the background scattering scales with the illumination area,high-NA objective helps the SNR by both increasing the snal ~collecting more light! and by reducing background.

Emitted fluorescence and backscattered laser lightthen recollected and recollimated by the same microscobjective and pass through the dichroic beamsplitter.sidual laser light is then filtered out as described above,fluorescence is focused by the microscope tube lens throa pinhole aperture located at the microscope image plaThe pinhole serves to spatially reject out-of-focal-plane ligand gives improved axial resolution, known as the confoadvantage. The diameter of the pinhole determines the alute depth of field for a confocal image, and an excelletreatment of pinhole selection is given elsewhere.86 Note thatthe confocal advantage also helps with SMS, since a smadepth of the matrix surrounding the single molecule islowed to present photons to the detector, reducing baground. This can be especially important in biological sytems, where out-of-focus cell autofluorescence needs tominimized.

Due to the limited number of photons available fromsingle-molecule emitter, selecting the proper sized pinholvery important. Smaller pinholes give superior axial resotion at the expense of transmission through the pinhole,are more difficult to align. The magnification of the micro


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FIG. 5. ~Color! Near-field scanning optical microscopy~NSOM or SNOM!. ~a! Schematic of a representative experimental setup:~OF! pulled and metal-coated optical fiber representing a near-field source of illumination that is placed very close to the sample surface, (O) objective, (F) filters, ~SPAD! siliconphoton counting avalanche detector.~b! NSOM image of single molecules of R6G on a silica surface using laser excitation at 514 nm. Field of2.6mm32.7mm, peak signals represent;250 photocounts in 20 ms, and the gray scale signifies large signal~white! to low signal~black! ~from Ref. 91, usedby permission!.

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scope objective also influences the transmission throughconfocal pinhole, since the size of the image spot atpinhole is simply the diameter of the diffraction-limited lasspot multiplied by the objective magnification. For a 603

microscope objective, a 75mm diameter pinhole allows hightransmission (.90%), gives good axial resolutio(,200 nm), and is easily aligned with a simple three-amicrometer stage. Following the pinhole, the laser lightreimaged onto either a single element detector, such asSPAD described above, or it can be dispersed by a mochromator onto a CCD to obtain single-molecule emissspectra, a technique that will be detailed below.

A confocal image is created by raster scanningsample stage underneath the laser spot, typically witcomputer-controlled piezo-electric stage. For confocal SMthe resolution of the piezo stage should be better than 50and a closed-loop piezo scanner makes it much easielocate and remain positioned directly on top of a single mecule, but is not absolutely necessary. A typical confocalage of single nile red molecules embedded in a thin poly~m-ethylmethacrylate! ~PMMA! file is shown in Fig. 4~b!. Thetwo-dimensional~2D! image is 100 by 100 pixels, represening a 10mm310mm area, and is acquired in 100 s. Eapixel has a 10 ms dwell time and the total number of photcounted in that time are displayed in a colorized gray scranging from dark~fewer integrated photons! to light. Theimage was scanned horizontally from the top to bottom; seral of the nile red molecules showed fluctuations duringscanning; and others showed photobleaching as evidencethe spots that are cut off before the scan over the molecucomplete.


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2. Near-field scanning optical microscopy

NSOM is another scanning method that is used for SMFigure 5~a! shows a simplified schematic for a near-fiescanning microscope. Unlike the previously described ‘‘ffield’’ techniques, which are all governed by classical optinear-field optics can be used to generate a laser spot smthan the diffraction limit by using an aperture with a diameter much smaller than the wavelength of light and bytecting the light that leaks through this small hole. If thradiation that propagates through this small hole is detecin close proximity to this aperture, that is within an axidistance on the order of the aperture diameter~far below theoptical wavelength!, the spot size detected will be that of thsub-wavelength aperture. Of course, as the axial distafrom this aperture increases, the spot size increases rapreaching the diffraction limit in the far-field regime. The ideof near-field optics has been widely known for some timewas first described by Synge in 1928 and initially demostrated for microwave wavelengths in 1972~see Ref. 87!.

Near-field processes are characterized by low intenthroughput for light through these tiny apertures. In the eastudies, to create a relatively efficient aperture suitableoptical wavelengths, a single-mode optical fiber was heaand pulled until it fractured, forming a tip much smaller thathe original fiber core.88 The fiber is then coated on the sidewith thin film of a metal, such as Al, thus making a wavguide for the light with a tiny aperture at the end, whichtypically 70 nm in diameter. Other improvements in nefield tips have recently appeared; for a review see Ref.Using scanning technologies developed for probe techniqsuch as atomic force microscopy, the fiber can be helddistance;5 nm from the surface and scanned. Transmit


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light and fluorescence can then be collected in the far fieldtraditional optics, and the signal detected with a single ement detector, as with a confocal microscope.

The first SMS experiments at room temperature wperformed in 1993, using NSOM to image single DiIC12

molecules embedded in a thin polymer~polymethyl-methacrylate! film.90 Strikingly, the authors observed varyinfluorescence emission patterns that were indicative ofabsolute orientation of the DiIC12 molecules in the polymefilm and analyzed them by considering the interaction wthe inhomogeneous near-field electric field and the molectransition dipole. NSOM has also been used to record flrescence spectra of single molecules at room temperarevealing heterogeneity among the spectral propertiessingle emitters as a function of time,83 and has been used tmeasure single-molecule fluorescence lifetimes.91–94

The primary advantages of NSOM are:~a! the reducedsample volume that is offered by the reduced laser spot sgenerating lower background counts,~b! immediate orienta-tion information, and~c! the fact that monitoring the positioof the scanned fiber tip gives topographic information simtaneously with fluorescence images. However, NSOMseveral limitations, most notably that the pulled fiber tipsbrittle and are subject to breakage, especially over samwith high surface roughness, limiting this technique to flsamples. It is also not possible to image single moleculesfrom the sample surface, since the near-field regime quicdisappears in thez direction, preventing the use of NSOM imany biological systems, such as in a cell interior. Furtheven though many improvements have been made in pdesign, the typical coupling efficiency through a pulled fibis ;1025– 1026 and it is difficult to consistently make fiberwith the same aperture size. Finally, the modest increasspatial resolution comes with greatly increased experimecomplexity when compared to confocal microscopy. Aspotential solution to some of these issues, novel apertureNSOM techniques have been proposed, in which antrasharp tip is used as an antenna to localize the excitaregion, and this technique may find further utility in singlmolecule studies.95–98 Another approach to ultraresolutioinvolves stimulated emission depletion in the region encling the focal spot,99 but this method has not been appliedsingle molecules to date.

Figure 5~b! shows a typical NSOM image of R6G moecules on a cover slide over a 2.6mm32.7mm range.91 Thebright spots correspond to single chromophore fluoresceThe diameter of the spots is roughly indicative of the relution of the microscope, demonstrating lateral resolutslightly beyond the diffraction limit, a tradeoff that was main this particular experiment to obtain a higher count rfrom the molecules. In the white boxed region, the two-lobstructure at the right is the ‘‘image’’ of a single moleculeexpected due to polarization effects.76 The left molecule inthe box photobleached halfway through the time requiredscan over the molecule. The arrow shows a subsequentdifferent scan direction that was used to collect fluorescelifetime as a function of position, illustrating the strong aterations in excited state lifetime that occur due to intertions between the metal tip and the molecule.91


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B. Wide-field methods

Perhaps the simplest method to observe single-molefluorescence is to use wide-field microscopy. As the naimplies, a laser or arc lamp is used to illuminate an aseveral microns in diameter, as in a traditional microscoProper filtering is used to eliminate excitation light andpass the single molecule fluorescence to a two-dimensiarray detector, usually a CCD as described above, thoughbest single molecule emitters can even be observedone’s own properly dark-adapted eyes!~Not only can themolecules be seen with the unaided eye, the colors cansensed, as well as color changes and amplitude fluctuator blinking.! Performing SMS in this fashion has two majoadvantages: many individual chromophores can be obsesimultaneously, and the position of chromophores canmonitored at near video rates, allowing researchers toserve translation of single molecules in real time. The pmary limitation of wide-field microscopy methods is that thmaximum frame acquisition rates for CCD cameras is slowthan the response time for single element detectors, buexperiments requiring maximum temporal resolution onorder of 50 ms, wide-field methods have become a popand useful SMS technique.

Even though a large region of the sample is imaged othe detector image plane, the spatial resolution of wide-fitechniques is diffraction-limited if research-quality micrscope objectives are used. Thus, with high NA optics, sinmolecules will be imaged as spots on the CCD w;300 nm diameter~FWHM! referenced to the sample planHowever, it should be noted that with sufficient SNR itpossible to pinpoint the location of these spots to better tthe diffraction limit. The accuracy with which the emitterposition can be determined is controlled by the size ofCCD pixels and the overall magnification of the microscopSpecifically, for a CCD camera with 15mm315mm pixelsmounted on a microscope with a 1003 objective and a 53beam expander lens at the intermediate image plane~giving a5003 total microscope magnification!, a wide-field imagewould display a single molecule as a spot that is 10 pixelsdiameter, with a Gaussian intensity profile. The positionthe center of this spot can easily be known to the neapixel, or 30 nm in this case, a concept that is key in studydiffusion or other similar processes. Again, with sufficieSNR, by fitting the illuminated pixels to a Gaussian profithe center can be located to a position even small thannearest pixel. In principle, infinite spatial resolution canobtained~for one spot only; the limitations on the ability tresolve two spots still apply unless multiple colors are usto distinguish overlapping spots100! by dispersing the imageover a greater number of CCD pixels, either by using a Cwith smaller pixels, a larger pixel array, or by increasing tmagnification, but several factors prevent this. First, singmolecule photobleaching limits the total number of detecphotons, and one needs to have a sufficient number of ptons per pixel for a given integration time to maintainproper SNR. Second, CCD cameras typically have readnoise, which places limits on the degree to which the emitphotons can be spread over detector pixels. Finally, the re


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FIG. 6. ~Color! Epifluorescence microscopy.~a! Schematic of a representative experimental setup: (O) objective, (D) dichroic beamsplitter, (F) filters,~CCD! two-dimensional array detector.~b! White light transmission image of a single CHO cell; various organelles in the cell interior are weakly visibl~c!Epifluorescence image for a 100 ms integration time of the same cell, showing the emission from single Cy5-labeled MHCII complexes as descritext. Laser illumination at 633 nm provided an intensity of;5 kW/cm2 at the sample plane. The epifluorescence was collected with a 1003 magnification,1.3 NA, oil-immersion objective~CFI PlanFluor, Nikon, Burlingame, CA! and imaged through a 645 nm dichroic mirror and a 670 nm band-pass~Omega Optical Inc., Brattleboro, VT! on an intensified frame-transfer CCD camera~I-Pentamax, Roper Scientific, Trenton, NJ!. The SBR was 1.6. Theaverage SM signal without background was 7516206 counts, and the average background was 477677 counts.~From Ref. 103, used by permission.!

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There are two popular techniques for wide-field SMimaging: epifluorescence and total internal reflection~TIR!microscopy. We will discuss the implementation of eatechnique and the relative advantages and disadvantageach method below.

1. Epifluorescence

An epifluorescence SMS microscope can readily be cstructed from commonly available commercial microscopas epifluorescence has been used for many years for biocal applications in particular. This method was first applto room-temperature SMS in studies of biomolecumotors.101 Figure 6~a! shows a simplified diagram of an epfluorescence microscope for SMS. To illuminate an area seral microns in diameter, it is necessary to use an input bthat isnot collimated. A simple method to create a laser spof appropriate diameter is to focus the laser beam ontoback aperture of the microscope objective. This is most eily accomplished using a lens with a focal length loenough that the lens can be mounted externally to the miscope; a typical lens used has a 0.5 m focal length. The inlaser beam is then reflected toward the microscope objecby a dichroic beam splitter. The microscope objective shohave the highest magnification available for most wide fiapplications for the reasons discussed above. For all Sexperiments, the NA is also a key parameter. N5n* sinfmax, wheren is the refractive index of the medium


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between the sample and the objective andfmax is the maxi-mum collection angle of the objective. To maximize the dtected light, the NA should be as high as possible, and thusually accomplished by using an objective designed toimmersed in a high-n medium. For most SMS applicationsan oil immersion (n51.51) objective is preferred, and thesobjectives have an NA as high as 1.4. For imaging in sobiological environments, it may be favorable to implemenwater immersion (n51.33) objective, which usually haNA51.2, exchanging some light gathering ability for improved image quality, but for most SMS experiments, theobjective is preferable. Naturally, the oil used must not cotain fluorescent impurities. The flatness of field for the mcroscope objective also has a significant bearing on wfield imaging applications, especially if distance parametacross the image need to be carefully quantified. Microscobjectives designated ‘‘PlanApo’’ are designed with the higest flatness of field and color correction, and should be ufor all wide-field imaging. For imaging at short wavelengthit also may be necessary to choose a microscope objecwith very low fluorescence.

Fluorescence emission is collected back throughsame microscope objective and transmitted through thechroic beam splitter. Residual laser excitation light isjected by appropriate long pass filters and/or notch filtershighlighted earlier. The light is then focused via the micrscope tube lens to a CCD camera. If additional magnificatis desired, relay lens systems with magnifications 23 – 83are commercially available and are easily installed intomicroscope.

One of the applications of wide-field SMS is the studymotion of critical structures in the membrane of singcells.102 Single-molecule labels are preferred to multiplebels or large beads in order to minimize disruption of natstructures. One recent effort has explored diffusion of sin



FIG. 7. ~Color! Total internal reflection microscopy using a prism.~a! Schematic of a representative experimental setup: (P) prism, (O) objective, (F) filters,~CCD! two-dimensional array detector, with the emitting single molecule signified by a star.~b! Image of the fluorescence emission over a 2.4mm32.4mm region from single copies of the GFP mutant T203Y immobilized in PAA in the TIR microscope. Conditions:pH 7, 100 ms exposure, and 2 kW/cm2

of 488 nm pumping light at the gel-cover slip interface. Fluorescence was imaged with a Nikon Diaphot 200 inverted microsccope, 1003 1.4 NA PlanApooil-immersion objective, via a 515EFLP long-pass or 535DF55 band-pass filter~Omega Optical! on a frame-transfer, intensified CCD camera~PrincetonInstruments I-Pentamax! ~from Ref. 109, used by permission!.

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transmembrane proteins embedded in live cells.103 Briefly,diffusion of the Major Histocompatibility Complex of TypII ~MHCII !, an essential binding protein forT-cell recogni-tion, was studied to determine how these important protemove around in the cell membrane. Chinese hamster o~CHO! cells were selected because they adhere nicelyglass microscope coverslips and because the surface oppthe adhered side is extremely flat. Figure 6~b! shows an im-age of a CHO cell under white light illumination. The edgof the oblong cell are clearly seen in this image. To labelMHCII proteins in an unintrusive manner, a short polypetide that strongly binds to the MHCII is labeled with a singCy5 dye molecule and the cell is exposed to these protewhich then bind to the MHCII structure in the cell membrane. Figure 6~c! shows an epifluorescence image of tlabeled cell. Only dye labels at the surface away fromglass are visible because the depth of field in an epifluocence microscope is;300– 500 nm and the CHO cell is seeral microns thick. The authors were able to monitordiffusion of the MHCII proteins by tracking the positions othe Cy5 labels with 100 ms integration times.

2. Total internal reflection

TIR microscopy can also be used to observe single mecules with high sensitivity. TIR measurements make usethe exponential decay of the evanescent field generatedtotal internal reflection at a high-index to low-index bounary, most typically at the interface between a glass or qucoverslip and air or water. For angles of incidence measufrom the interface normal~u! above the critical angle, theevanescent field intensity (I ), ~which is proportional to the


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A TIR microscope can be implemented by couplingmicroscope coverslip to a prism with an index matching flu(n51.51 for glass!. A laser beam can easily be coupled inthe prism above the critical angle to achieve TIR, andmicroscope can image the fluorescence excited from thenescent field created at the interface. Figure 7~a! shows aschematic drawing of a prism-type TIR setup used to imasingle dyes106 and single copies of GFPs embedded in



FIG. 8. ~Color! Total internal reflection microscopy, through-the-objective configuration.~a! Schematic of a representative experimental setup: (O) objective,(D) dichroic beamsplitter, (F) filters, ~CCD! two-dimensional array detector.~b! Example image of R6G in PMMA, scale bar 10mm, incident intensity at532 nm50.56 kW/cm2. This image was recorded with a TE300 inverted microscope~Nikon! with a 603, 1.4 NA oil-immersion objective, 545LP dichroicfilter, HQ525/50 emission filter~Chroma, Brattleboro, VT!, and a frame-transfer, intensified CCD camera with 100 ms integration time~Princeton InstrumentsI-Pentamax! ~from Ref. 110, used by permission!.

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water-filled poly~acrylamide! ~PAA! gel matrix to restrictBrownian diffusion;107 see Refs. 108 and 109 for morsample preparation information. For this experiment, illumnation was provided by the evanescent wave generateTIR of a linearly polarized laser beam at the interfacetween the quartz cover slip and the gel sample. The corincidence angle for TIR on this interface was obtained byuse of a triangular quartz prism and a layer of glycerol onof the upper cover slip. A quartz prism was used insteadglass to minimize background autofluorescence. Typicalcitation intensities were of the order of 1 kW/cm2, but ofcourse, the distance of the molecules from the interfacenot controlled, so the absolute intensity at each moleccould vary by as much as a factor of 2 or more. In typicmeasurements, saturation of the emission was avoided.ure 7~b! shows a rendered three-dimensional plot of Gfluorescence as a function of two-dimensional position inmicroscope plane; it should be noted that this type of ployet another method to display single-molecule fluoresceimage data, and it is easy to compare the relative intensof individual molecules by examining relative peak heigh

TIR can also be created by coupling an input laser bethrough the extreme edge of a high-NA microscope objtive, in order to place the evanescent field at the positionthe first boundary between a glass cover slip and a lowindex medium. The method is then called through-thobjective TIR, a technique first implemented for SMSFunatsuet al.101 Figure 8~a! shows the through-the-objectivTIR microscope. As with the epifluorescence microscope,input laser beam is not collimated, and identical optics cbe used for both setups, by simply mounting the final mirof the laser input path on a translation stage. The microsc


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can be switched between epifluorescence and TIR by simtranslating this mirror such that the input beam is coupthrough the edge of the microscope objective or throughcenter as described in detail in Ref. 110. Figure 8~b! showsan image of single R6G molecules embedded in a PMMfilm.

The primary benefit of TIR is the reduced backgrouthat comes from the reduced illuminated sample thicknand the lower absolute powers that are required to illuminthe samples. However, TIR is usually limited in its useregions located within;150 nm of the glass microscopcoverslip, and for imaging away from the glass coversinterface, such as the MHCII diffusion study describabove, epifluorescence can be useful. In one report, TIRthe interface between the cell and the surrounding aquemedium has been described.111 Several detailed comparisonof the various TIR configurations have been presented.110,112

IV. SPECIFIC DETECTION MODALITIES

Since the inception of SMS over 10 yr ago, great effohave been made to extract the maximum information frevery photon emitted from a single fluorophore, often a dficult task due to the intrinsically small signals inherentsingle-molecule fluorescence. The ability to observe invidual members of an ensemble population has demonstrthat significant heterogeneity exists even among moleculecrystals. SMS has shown that molecules are very sensitivtheir local environment, and this sensitivity can be exploitto gain new insights into a variety of processes. Numertechniques have been developed that can characterize sephysical properties, such as molecular orientations, ma


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molecular conformational changes, energy transfer pcesses, and even providing insight into the physics of flrescence itself. The overriding theme is to obtain as minformation from the emitted photons as possible by recoing their polarization, time of arrival, wavelength, actiospectrum, etc. To study diffusion effects, the position ofmolecule is recorded as a function of time and the dataanalyzed for departures from Brownian motion.102,103,113Thissection briefly introduces several useful SMS detection mdalities, describes the general implementation of the conchighlights a few recent achievements using each respectechnique, and directs the interested reader to detailedticles available in the literature.

A. Polarization microscopy

Despite the fact that the spatial resolution of~diffraction-limited! optical methods is on the order of a few hundrnanometers, the extreme sensitivity of SMS to changelocal orientation allows one to probe effects on the molecuscale. One of the most powerful SMS techniques curreemployed is polarization microscopy, which allows onedetermine the exact angular orientation of single molecuIn most cases, the absorption and emission dipoles of higfluorescent molecules are parallel, and this is assumedfor simplicity. One way to extract orientational informatiois via modulation of the excitation polarization@polarizationmodulation,~PM!#. The physical basis for PM is simple; thexcitation of a molecular transition is dependent on thegular alignment of the excitation electric field vectorE ~po-larization! and the molecular transition dipolem, specificallythe probability of excitation depends uponum"Eu2. For asingle molecule, there is only a single transition dipoleconsider, so there is a discrete polarization anisotropy offluorescence. If the molecule is static or rotating slowly wrespect to the integration time, then one can observe a2

dependence of the fluorescence signal as the polarizatiothe excitation laser beam is rotated with respect tom. Similararguments apply to the emission polarization: becausemolecular emission has the shape of the dipole emissiontern, detection of the emission in two orthogonal polarizatchannels allows extraction of information about the molelar orientation. For reviews of polarization microscopysingle molecules, see Refs. 114 and 115.

Excitation polarization modulation can easily be incoporated into a confocal or wide-field microscope by usingelectro-optic modulator to rotate the polarization of a lineapolarized input laser beam.116 Confocal PM geometries havbeen used to measure reorientations of dye moleculepolymer films117 and hindered rotation of dye moleculelinked to tethered DNA molecules in an aqueoenvironment.118 Wide-field PM experiments have been usto observe molecular machines in real time, includingrotation of F1-ATPase,119 and to study the rotational flexibility of single kinesin motor proteins as a function of nucotide state, revealing a previously unseen highly mobile fowhen adenosine diphosphate~ADP! is bound, providing newinsight into the complex walking mechanism for this impotant protein.120,121


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As an illustration of the use of PM to extract orientatioinformation for a single molecule in an epifluorescenceperiment, Fig. 9 shows results for a singly labeled poly~buta-diene! chain in a PMMA matrix.50 The pump polarizationwas electro-optically toggled between two orthogonal traverse directions in the sample plane, denotedX andY. Theemitted fluorescence was collected through appropriateters as usual, but with no analysis of the polarization. Feach pump polarization, a 100 ms image of the single mecules was obtained. After the end of the experiment,time trajectory of each of the molecules in the field wextracted, and data from a typical molecule is shown in F9~a!. The ratio between the two emission signals cleachanges as a result of rotation of the molecule, up untilpoint of photobleaching at;22 s. Figure 9~b! shows oneway to display the data as a polar plot of in-plane angle wthe radial position showing the total intensity of the molecuestimated using (X21Y2)(1/2). From molecule to moleculethe dynamical character of such traces can change dramcally as a result of the hidden heterogeneity in an amorphmaterial.

An alternate approach to extracting polarization informtion involves pumping with circularly polarized light anresolving the emission into two orthogonal directionsA andB.122 In this case, the fluorescence polarization is usuacalculated using (I A2I B)/(I A1I B). If the molecules are ro-tating rapidly, the emission will be unpolarized and the dtected P values will cluster around zero. However, if throtation is slow, characteristic departures fromP50 will beobserved. Some investigators have done both: PM as weresolution of the emission, which can extract additionalformation such as the cone angle of a covalently attacfluorophore.115

It should be noted that TIR and NSOM geometries cintroduce complicated polarization effects that can be usefurther probe molecular orientations. In the case of Tp-polarized input laser light produces evanescent polartion in both the transverse and axial (z) directions, the latterof which can be used to excite molecular dipoles orientedthez-axis direction perpendicular to the plane of the samp

FIG. 9. Example of the use of polarization modulation microscopy to sethe in-plane orientation of the transition dipole moment of a single fluophore on a single polymer chain.~a! Fluorescence emission withx and ypolarized pumping as a function of time, illustrating a continuously chaing in-plane dipole angle.~b! The trajectory of the in-plane dipole angle anthe overall brightness of the molecule shown in~a!, which shows the rangeof angles sampled by the dipole. The open squares and filled circles cspond to the early half and last half of the trajectory, respectively~from Ref.50, used by permission!.


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A prism TIR geometry was used to image the emissionpole orientation for single molecules by observing the emsion patterns for single molecules,123 capitalizing on spheri-cal aberrations present when imaging several microns afrom the glass cover slip with high-NA microscope objetives. This effect caused thez-oriented molecules to appeaslightly defocused, giving them a ‘‘doughnut’’ emission patern, whereas molecules with the transition dipoles alignethex–y plane had a normal emission pattern. The local eltric fields vary wildly across the aperture of an NSOM fibprobe, due to near-field effects described elsewhere.76 Be-cause the aperture is orders of magnitude larger than a smolecule, a molecule interacts with different local electfields as the fiber is scanned across the molecule. Thetected fluorescence intensity maps how the molecule inacts with these complex fields, and this can be used to pthe orientation of molecules, an effect originally seen infirst NSOM SMS experiments.76 Other methods that havbeen to extract orientation involve annular illumination124

and simultaneous resolution of the emission into three poization directions.125

B. Spectral dispersion of emission

The previously mentioned single-molecule detectmethods involve counting the total numbers of photons obroad-band detector, usually a CCD camera or a SPAD, msuring total redshifted fluorescence as a function of time.also possible to obtain spectral information about singmolecule emitters as a function of time by using a modifiversion of the scanning SMS microscopes described abSpecifically, the single element detector can be replaceda grating spectrometer to spectrally disperse fluorescencelowed by a CCD camera placed at the slit position ofgrating spectrometer. Each vertical strip of pixels onCCD chip can be summed to create a ‘‘superpixel’’ that reresents a single wavelength, and a complete fluorescspectrum is then obtained with each frame read out onCCD. One needs to realize that the emission spectrum frosingle molecule has a SNR that is inherently worse thawavelength-integrated signal, since the same number of ptons is now being dispersed over many superpixels, whbehave like individual detectors. Further, dispersion opintroduce additional optical losses in the microscope systFor this reason, high resolution grating spectrometers aredom used in favor of low f-number systems with highthroughput.

The original single-molecule emission spectra weretained for molecules embedded in solids at cryogetemperatures,80,81 a topic covered in several reviews.82,126

Such spectra are actually equivalent to resonance Raspectra, and the line positions show the Raman active moof the electronic ground state. The primary benefit of woing at low temperatures is that linewidths are extremely nrow, and it is easy to see small changes in the positionthese sharp peaks. Further, even very weak lines are edetected in the single-molecule spectrum since the phofor a given spectral feature are concentrated on a small nber of pixels. Spectra have been obtained for single m


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ecules embedded in a variety of crystalline127 andpolymer80,81 hosts, revealing quite distinct spectral line potions arising from different local environments.

Emission spectra have also been acquired for single mecules at room temperature, revealing similar effects tolow temperature studies, including spectral diffusion and herogeneity among the peak fluorescence positions for difent molecules83,84,92and for extended chromophores in singpolymer chains.128 The primary difficulty in obtaining roomtemperature spectra is that linewidths are quite broad, tcally on the order of 75 nm~FWHM! or more, making itchallenging to trade off SNR and spectral resolution. Acent study129 observed detailed variations in the local chaacteristics of thin polymer films by recording spectra frosingle nile red molecules, a polarity-sensitive dye ablesense charge transfer with the surrounding medium. Thethors took spectra of nile red in relatively nonpolar PMMand polar poly~vinyl alcohol! ~PVA! films, measuring theposition and widths of the fluorescence spectra. Interestintwo distinct peak widths were observed for moleculesPMMA films, an effect the authors attribute to the presenof two domains in the polymer film with different local rigidities. Conversely, the PVA films showed a broad distribtion of environments whose rigidity strongly dependedthe level of hydration.

An example of fluorescence emission spectra frsingle molecules at room temperature is shown in Fig.Single DiIC18 molecules embedded in a PMMA film werilluminated with 1.5 kW/cm2 532 nm laser light in a confocamicroscope configuration. The top and middle panels shspectra from two different single molecules, with an integtion time of 30 s. One may note how the individual moecules display radically different spectra, a result of differelocal interactions. For comparison, the lower panel of Fig.shows a bulk spectrum taken with a 1 sintegration time andgreatly attenuated laser intensity for a film of many thosands of DiIC18 molecules in PMMA. The ripples in the bulkspectrum are a result of the transmission characteristicthe microscope filters~see Fig. 2!.

FIG. 10. A fluorescence spectrum of a single DiIC12 molecule embedded ina PMMA film, recorded with the filtering described in Fig. 4, but obtainby directing the emission through a grating spectrometer to which a liqnitrogen cooled CCD array detector has been attached. The excitation isity was 1.5 kW/cm2 at 532 nm.~Top and middle panels! Two differentsingle molecules, with an integration time of 30 s.~Lower panel! Bulkspectrum, 1 s integration time, greatly attenuated laser intensity.


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C. Fluorescence resonant energy transfer „FRET…

Another often-used fluorescence technique is FRwhere a short-wavelength ‘‘donor’’ fluorophore is pumpby a light source, and its energy is transferred to a lonwavelength ‘‘acceptor’’ molecule via Fo¨rster dipole-dipoleinteractions, whereupon the acceptor fluoresces, emitlight at a long wavelength. The FRET efficiency,Ed , is de-pendent on the distance between the donor and acceptorcan be expressed:Ed51/$11(R/R0)6%, whereR is the dis-tance between the donor and acceptor molecules, andR0 isthe characteristic distance where 50% of the energy is trferred from the donor to the acceptor. Under appropriate cditions, the energy transfer efficiency may be extracted frthe ratio of the emission intensity of the donor to the emsion intensity of the acceptor. Molecular factors that inflenceR0 are the spectral overlap of the donor–acceptor pthe refractive index of the medium, the quantum yields ofdonor and acceptor, and the relative orientation of the trsition dipoles for the donor and acceptor; typicalR0 valuesrange from 2 to 6 nm. Excellent treatments of FRET aavailable in several monographs and reviews,130–132 andsingle-molecule FRET has been the topic of a recreview.133

As a result of the sixth power dependence ofEd on thedonor–acceptor distance, FRET is widely used to meassmall distances between sites on large molecules sucproteins, and has been a useful bulk fluorescence assaquite some time.134 The spatial scale of FRET is far belothe diffraction limit, so it is in principle possible to extrachigh resolution distance information in this manner. Rcently, FRET has shown to be a powerful tool for using SMto study conformational changes of biological macromecules. Haet al.135 were the first to use FRET to detect coformational changes of a three-helix ribonucleic acid~RNA!junction, whose structure folds upon binding of the ribosmal protein S15. In this experiment, the authors used a cfocal microscope, but split the detected light to two SPAdetectors using a dichroic beam splitter; the transmitted~acceptor! intensity and the reflected blue~donor! intensitycould then be monitored simultaneously.

One issue that should be considered with FRETsingle molecules is the fact that the degree of energy tranis extremely sensitive to the fluorescence properties of edye molecule. Fluorescence dynamics can be extremcomplicated for even the simplest systems, as shown abfor the spectral diffusion of single molecules. Fluctuationsthe fluorescence properties of the dyes themselves duvarying interactions between the dye and its local envirment can drastically change parameters such as the emispectrum and quantum yield, and these two propertieshave to be measured separately in control experiments.ther, the rotational characteristics of the dye labels affectorientational factor inR0 , and these can dynamically changon the time scale of the desired experiment. Since~for a fixeddistanceR) the FRET efficiency goes from a maximuwhen the donor and acceptor transition dipoles are aligparallel to zero when the dipoles are perpendicular,could mistake a conformational change for a rotational ev


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if the relative dye rotation is slow enough. While these cocerns are less of an issue with bulk measurements sinceensemble is a statistical average of all orientations, quanyields, and other relevant fluorescence characteristics,needs to perform many controls with a single-molecule stem. Even small changes in theR0 value will have hugechanges in the FRET efficiency. For example, the rotatiocorrelation times of the two dyes may need to be separameasured with polarization anisotropy to extract preciseformation.

Another limitation of FRET is that two separate dymolecules must be introduced to the system. Becauseprimary benefit of SMS is its inherent sensitivity to systeheterogeneity, the two dye labels must be carefully intduced to the identical location of all studied molecules, afor proteins, the specific introduction of multiple dye labeis a challenging problem,136 especially for those located inside living cells. However, fusions to GFP avoid this difculty because the DNA that codes the protein for studybe genetically modified to include the DNA for GFP atspecific location in the DNA sequence, thereby creatingnaturally fluorescent label at a known location. A recentfort in the Moerner lab137 used a so-called ‘‘cameleon’’ construct that is a fusion of two GFP mutants to thCa21-dependent calmodulin protein.138 Upon binding Ca21,the cameleon protein contracts, pulling its two ends clotogether. On one end of the cameleon, a blue-shifted varGFP is expressed, and on the opposing end a redshiftedmutant is expressed. The authors pumped the blue doGFP and were able to detect a significant increase in FRefficiency~to the red acceptor GFP! upon Ca21 binding, thefirst example of a single-molecule FRET system withouting dye labels. However, the poor photostability of GFPduced the amount of information obtainable.

In spite of these issues, single-molecule FRET continto be a useful tool to study biomolecular dynamics. To adress the orientational factor, often the two fluorophoresattached with floppy saturated alkane linkers to reducerotational correlation time. In addition, rather than atteming to extract precise values of the interdye distanceR, largeand qualitative changes in FRET efficiency are sufficientconclude that the conformation of the system has changeaddition to the studies mentioned above, single-molecFRET has been used to study enzyme conformatiodynamics,139 the folding of individual RNA enzymes,140

single GCN4 proteins,141 DNA unwinding by Rephelicase,142 and the list of experiments continues to grow.

D. Two-photon excitation

Recently, two-photon excitation of fluorescence hascome a powerful tool for imaging biological systems in thrdimensions, due to the quadratic dependence of two-phoexcitation probability on laser intensity, a process that creaa vastly reduced excitation volume.143 This aspect of two-photon microscopy can be thought of as an enhancementhe confocal advantage, where the depth of field is extremthin, since the laser intensity outside the beam waist is mlower than that at the laser focus, allowing for superior cro


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sectioning ability. Indeed, the construction of a two-photmicroscope is very similar to a confocal microscope usinfemtosecond pulsed laser excitation source, except thaconfocal pinhole is removed, since it is no longer needThe primary advantages of two-photon microscopy are twfold. First, the reduced sample volume excites fewer baground impurities, lowering background fluorescence. Sond, it is possible to access transitions in the near UV usnear infrared~IR! light, a powerful benefit since many materials fluoresce strongly when pumped in the near UVare transparent to IR light, including cells and even the glused in optical elements.

Two-photon single-molecule fluorescence was firstported in 1995 for single Rhodamine B molecules diffusithrough the reduced two-photon laser focus.144 Immobilizedsingle molecules have also been observed with a comparSBR to conventional confocal microscopy, but the probaity of photobleaching was increased by approximately a ftor of 2, possibly due to absorption of a photon while tmolecule is in a dark triplet state,145 a possible limitationinherent to this technique. There is also the possibilityheating of the host matrix or liquid by overtone absorptioOne must also take great care when focusing femtosecpulsed light in a two-photon microscope; even modest poers can generate white light in the sample, limiting the intsity regime accessible to SMS experiments, which oftenquire relatively high excitation rates to obtain an adequSNR. Nonetheless, with the recent commercial availabilityfemtosecond pulsed lasers, the use of two-photon miccopy will continue to grow, especially for use in cellulasystems and other thick samples that are subject to autorescence.

E. Time-dependent dynamical studies

No matter which experimental variable is measured,time-dependent changes in the signal are usually of ginterest. In fact, it is a particularly important advantagesingle-molecule optical spectroscopy that the system unstudy can often be followed noninvasively as it proceethrough different time-dependent states. This dynamicalformation is an example of the type of information that is neasily available from ensemble averaged-measurementsas x-ray crystal structures or cryo-electron microscopy,example. To be more specific, if a bulk ensemble samplcomposed of enzymes in different chemical states, the stare often not synchronized and details of the state chancan be smeared out. However, a single-molecule experimthat follows the time-dependent behavior of each single mecule, one at a time, can remove this dynamic heterogenwithout synchronization.

Time-dependent effects can arise on many different tscales, from nanoseconds to thousands of seconds, andmay arise from a variety of factors. Here the time scalesphysical effects will be described proceeding from the fasto the slowest, and examples of the type of analysis in eregime will be briefly mentioned.


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1. Fluorescence lifetime measurements

On the shortest time scale of nanoseconds, the streaphotons emitted by the molecule contains information abthe system encoded in the arrival times of the individuphotons. One way to obtain information in this regime ivolves pumping the sample with short pulses from a reptively pulsed source such as a mode-locked laser or a pulight emitting diode. After each excitation pulse, at most onone photon can be emitted since only one moleculepumped. For sufficiently short excitation pulses, measument of the distribution of time delays between the pumphoton and the emitted photon allows a direct measuremof the excited state lifetime of the moleculetF . Once asingle molecule is microscopically selected by any of toptical configurations shown above, the lifetime can be msured by the standard technique of time-correlated sinphoton counting. In this method, digital timing electroniare used to measure the histogram of time delays for a spump photon–emitted photon pairs. For example, a specized personal computer board~TimeHarp, Picoquant! can bedriven by pulses signifying the time of the pump pulse athe time of detection of an emitted photon. Typically, tnumber of photons that must be detected to allow deternation of the excited state lifetime is on the order of 10with more photons required if high accuracy is needed.actual experiments, the time resolution of the measuremelimited in the usual way by the instrument response functiand the maximum repetition rate is limited by the dead tiof the detector. For the latter parameter, the SPAD detectypically have larger dead times than PMTs, but thereother advantages of the SPAD detectors as described ab

The earliest single-molecule lifetime measurementvolved flowing stream studies,146 followed the next year bylow-temperature single-molecule lifetime experimentsthe canonical pentacene/p-terphenyl system.147 The firstroom temperature studies observing the same moleculean extended time were performed on R6G on surfaces uNSOM methods,83,91,93 and a puzzling effect was reportethat the observed lifetime depended upon the exact posof the tip with respect to the molecule. This turned out todue to the quenching influence of the metallized portionthe tip on the molecular dipole emission. Later experimehave used far-field methods to avoid distortion of the oserved lifetime.92 In recent work, sophisticated data procesing methods have been devised that allow one to dynacally shift the time frame over which the lifetime imeasured, which allows observation of time-dependchanges in the lifetime due to dynamical quenching effearising from conformational changes.148 Single-moleculelifetime measurements have been useful in exploring tRdynamics,149 DNA conformational fluctuations,150 and elec-tron transfer at interfaces,151 to cite a selection of recent studies.

2. Single-molecule quantum optics

A further set of interesting time-dependent effects arifrom the fact that the single molecule is a quantumechanical object, and hence quantum-optical correlati


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are contained in the statistics of the photon emission timeven for cw illumination. On the time scale of the excitstate lifetime, photons emitted from single quantum systare expected to show photon antibunching, which meansthe photons ‘‘space themselves out in time,’’ that is, the prability for two photons to arrive at the detector at the satime is small. Antibunching is fundamentally measuredcomputing the second-order correlation of the electric fig(2)(t) @which is simply the normalized form of thintensity-intensity correlation functionCs(t)], which showsa drop below the uncorrelated value of unity when anbunching is present.152 For a single molecule, antibunchingeasy to understand as the individual molecule cannot etwo photons at the same time. After photon emission, a ton the order of the inverse of the Rabi frequency must elabefore the probability of emission of a second photon ispreciable. At sufficiently high laser intensity, Rabi oscilltions can be observed as the laser coherently drives the smolecule into and out of the excited state before emissoccurs.

In actual practice, in order to overcome time limitatiocaused by the dead time of photomultipliers or SPADs, tidentical detectors are used to measure the distributiontime delays between the arrival times of consecutive pairemitted photons as in the classical Hanbury Brown–Twexperiment.152 The expected antibunching in single-molecuemission was first observed for a single molecule of pencene in p-terphenyl153 demonstrating that quantum optics eperiments can be performed in solids and for molecules.course, if more than one molecule is emitting, the antibuning effect as well as any bunching effect due to bottlenestates both quickly disappear since the various resonantecules emit independently. The observation of high-contantibunching is strong proof that the spectral featuresindeed those of single molecules. A variety of othquantum-optical experiments have been performed on simolecules at low temperatures, including ac Stark effectselectro-optical mixing, reviewed in Ref. 126.

In the room temperature regime, an extremely staemitter is needed to complete useful single-molecule qutum optics experiments. For example, single CdSe/Cnanocrystals show antibunching on the short~ns! time scaleof the excited state lifetime,59 in spite of serious blinking andfluctuation effects on longer time scales.61 Surprisingly, re-searchers have shown that terrylene molecules protefrom photo-oxidation in a p-terphenyl crystal at room teperature show a very low probability of photobleaching48

and this system showed clear high-contrast antibunchin154

under cw illumination.Terrylene in p-terphenyl was recently utilized to produ

a high efficiency, highly quantum mechanical sourcesingle photons on demand.155 A high performance, roomtemperature source of single photons at a designated retion rate would be a key component in an optical quantcryptography and communication system. It was possibleuse a standard optical microscope and a simple dopedlecular crystal to make a source in which the probabilitysingle-photon emission per pump pulse was as large as 8a highly nonclassical value which strongly departs from


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usual Poisson statistics. With this stable emitter, single ptons could be emitted at a rate of several MHz for a total109 photons or more.

3. Intermediate time regime

In the time regime of microseconds to milliseconds,large array of dynamical effects can be felt in the singmolecule emission, such as photophysical effects like insystem crossing and photochemical effects such as prottion, electron transfer, or conformational fluctuationAutocorrelation analysis has long been recognized as aful method for statistical study of stochastic dynamical pcesses that may be partly obscured by noise.23,156By defini-tion, the autocorrelation measures the similarity~overlap!between the photon emission signalS(t) and a copy ofS(t)delayed in time by a lag timet

CS~t!5E S~ t !S~ t1t!dt. ~13!

The shape of the autocorrelation function containstails of the dynamical process, under the assumption thatdynamical process is stationary, that is, the dynamics mnot change during the time needed to record enough pharrivals to generate a valid autocorrelation. Because the ncomponent of the signal is uncorrelated, its contributionCs(t) decays quickly, leaving information about the~aver-age! time-domain correlations ofS(t). In practice, whenphoton counting is used, a commercial digital correla~Brookhaven, Malvern! is employed to measureCs(t) bykeeping track of the arrival times of photons. Modern coelators can do this over a huge logarithmic time scale coving many decades in time, a feature that is very usefulstudying the dispersive dynamics characteristic of amphous systems.

It is important to distinguish two regimes:~a! measure-ment on the same single molecule for as long as possand ~b! measurement of many single molecules, one atime, but summing together the signals from all of themincrease the SNR of the correlation function. The lattergime is the FCS limit mentioned at the beginning of Sec.and FCS is generally performed for emitters at very loconcentration in solution where diffusion is used to brieach new molecule into the volume to generate a new bof emitted photons. The former regime is the primary focof this article. The decay in the autocorrelation of the emitphotons for a single molecule of pentacene in p-terphedue triplet intersystem crossing effects was first reportedthe low temperature studies,3 and similar experiments inpolymer systems allowed identification of different typestwo-level systems in the host.157

4. Milliseconds and longer

Most single-molecule microscopy experiments explothe time scale from ms to many seconds, chiefly becausehigh intensity needed to obtain short-time information oftresults in premature bleaching. Moreover, most systems sied to date have shown some form of fluctuating, flickerinor stochastic behavior6 arising from photochemistry, spectra


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shifts, enzymatic activity,53 or environmental fluctuations between the chromophore and the surrounding host matrix.full array of physical effects leading to fluctuations will nbe reviewed here, but to give an example, Fig. 11 showstime dependence of the photon emission signal for a sincopy of a mutant GFP-even though the laser is on contously throughout the trace, the emission from the single ptein emitter turns on and off in a roughly digital fashion, bwith several characteristic time scales.109

Fluctuating time-dependent signals like those shownFig. 11 may be analyzed in several ways. First of all, itimportant to try to obtain as long a time trace with as ma‘‘on/off’’ switching events as possible to improve the stattical sample. If the time scale of interest is very long, thproper selection of the illumination duty cycle can be etremely helpful. With a sufficiently long time trace, calcultion of the correlation function is a useful first step; if thprocess is characterized by simple two-state kinetics, therelation function will be exponential. However, there are seral single-molecule experiments where the decays aresimple single exponentials, indicating more complkinetics.149,158 One example would be the behavior of thangular orientation of single probe molecules in a polymnear the glass transition,72 where stretched exponential kineics have been observed. Another would be the dark tdistributions of single CdSe quantum dots which showpower law time dependence.61 In the event that the signaseems to switch between two values, experimenters geally attempt to extract separate distributions of on timesoff times from the single-molecule time traces.122 When thisis possible, the experimenter can turn to the large arraymathematical analysis methods that have been previouslyveloped for the analysis of single ion channel data.14 As afurther example at room temperature, correlation analysisbeen applied to explore rotational correlations for molecuin polymers.159

For the case of single copies of the enzyme cholestoxidase, higher-order correlation analysis, in whichlengths of successive on-times were correlated, prouseful.53,160 By this method, evidence was gathered for thypothesis of fluctuating rate constants, an interesting typdynamical disorder only observable at the single-moleclevel.

V. DISCUSSION

This article has described in detail the experimental csiderations and microscopic configurations needed to

FIG. 11. Time trajectory of the emission from a single copy of the Gmutant 10C in PAA using a confocal microscope, with 10 ms integrattime and 514 nm pumping with an excitation intensity of 1.6 kW/cm2 ~fromRef. 109, used by permission!.


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serve single-molecule fluorescence. The SNR depends ua variety of properties of both the molecule of interest athe host matrix that should be carefully optimized. Micrscope configurations of both the wide field and the scanntype can be used for such measurements. In all casesextraction of the maximum amount of information from eaemitted photon is essential, and a variety of detection mdalities are available. With the large array of successful teniques described here, it is to be expected that a furtherpansion of single-molecule optical measuremenunobscured by ensemble averaging, will occur.

ACKNOWLEDGMENTS

The authors thank W. P. Ambrose, S. Nishimura, M. Vljic, and K. Willets for providing example data. This worhas been supported in part by the National Science Foution Grant Nos. 9816947 and 0212503. D. F. acknowledthe support of a NSF Graduate Fellowship.

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