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Single-Molecule Fluorescence Detection

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Single-Molecule Fluorescence Detection Proc. Nadl. Acad. Sci. USA Vol. 86, pp. 4087-4091, June 1989 Biophysics Single-molecule fluorescence detection: Autocorrelation criterion and experimental realization with phycoerythrin (laser-induced fluorescence/femtomolar sensitivity/fluorescence assays/phycoerythrin oligomers) KONAN PECK*, LUBERT STRYERt, ALEXANDER N. GLAZERt, AND RICHARD A. MATHIES* *Department of Chemistry, University of California, Berkeley, CA 94720; tDepartment of Cell Biology, Stanford University School of Medicine, Stanford, CA 94305; and tDepartment of Microbiology and Immunology, University of California, Berkeley, CA 94720 Contributed by Lubert Stryer, February 10, 1989 ABSTRACT A theory for single-molecule fluorescence ofindividual molecules. In this paper, we present a theory for detection is developed and then used to analyze data from single-molecule fluorescence detection and apply it in de- subpicomolar solutions of B-phycoerythrin (PE). The distri- tecting phycoerythrin at concentrations as low as 1 fM. bution of detected counts is the convolution of a Poissonian continuous background with bursts arising from the passage of individual fluorophores through the focused laser beam. The THEORY autocorrelation function reveals single-molecule events and Consider a solution of fluorophores flowing through a fo- provides a criterion for optimizing experimental parameters. cused laser beam. The emission from the illuminated volume The transit time of fluorescent molecules through the 120-fl consists of bursts of fluorescence from molecules passing imaged volume was 800 ps. The optimal laser power (32 mW through the beam superimposed on a continuous background at 514.5 nm) gave an incident intensity of 1.8 x 1023 due to Rayleigh and Raman scattering from the solvent (Fig. photons-cm 2.S-, corresponding to a mean time of 1.1 ns lA). The mean rate kb (photons s-1) ofbackground scattering between absorptions. The mean incremental count rate was 1.5 by the solvent is per 100 jts for PE monomers and 3.0 for PE dimers above a background count rate of 1.0. The distribution of counts and kb = O-J, [1] the autocorrelation function for 200 fM monomer and 100 fM dimer demonstrate that single-molecule detection was and the mean rate kf (photons s-1) of fluorescence emission achieved. At this concentration, the mean occupancy was 0.014 by a molecule in the beam assuming nonsaturating excitation monomer molecules in the probed volume. A hard-wired conditions is version of this detection system was used to measure the concentration of PE down to 1 fM. This single-molecule kf = o5IQ = 3.8 x 10-21 eQP/(rcW2). [2] counter is 3 orders of magnitude more sensitive than conven- tional fluorescence detection systems. Here I is the light intensity (photons cm-2 s-1), ob is the sum of the Rayleigh and Raman scattering cross sections of the Fluorescence spectroscopy is an ideal method for quantitat- solvent, oa is the absorption cross section of the fluorescent ing the concentration and location of fluorescent molecules molecule, Q is the quantum yield of fluorescence, E is the because of its high sensitivity. Fluorescence detection is extinction coefficient, c) is the beam l/e2 radius, and P is the widely used in immunoassay, flow cytometry, and chromato- laser power (photons s'). Fluorescence emission by a mol- graphic analysis, where the detection limits are from 103 to ecule is ultimately terminated by photodestruction (8, 13). 106 fluorescent molecules (1, 2), and in automated DNA The rate of photodestruction kd (S 1) is sequence analysis, where the detection limits are 106-107 molecules (3, 4). The development of more sensitive fluo- kd = aI(D9 [3] rescence detection systems is important because it would permit new applications of this technique in analytical chem- where ID is the quantum yield ofphotodestruction. The mean istry, biology, and medicine. lifetime td of a fluorescent molecule in the beam is 1/kd. In the quest for enhanced sensitivity, Hirschfeld (5) used The number ofdetected counts ns in a time interval At is the evanescent-wave excitation to detect an antibody molecule sum of nb, the counts from solvent scattering, and nf, the labeled with 80 fluorescein molecules adsorbed on a glass counts from fluorescence emission. The mean number of slide. Using a flowing sample, Dovichi et al. (6) achieved a background counts ,b is given by detection limit of 22,000 rhodamine 6G molecules in a 1-s integration time, and Nguyen et al. (7) extended this limit to PUb = kbAtEb, [4] 800 molecules with hydrodynamically focused flows. Mathies and Stryer (8) pointed out the limits imposed by where Eb is the collection efficiency from solvent scattering. photodestruction and detected three molecules of B- The probability Pb of havingj counts of background scatter- phycoerythrin (PE) in a probe volume of 10 pL. PE is an ing in a time interval At is given by the Poisson distribution: attractive fluorophore for enhancing sensitivity because ofits Abe high absorption coefficient, near unity fluorescence quantum Pb(j) = [5] yield, and large emission Stokes shift (9-11). Recently, Nguyen et al. (12) observed bursts of fluorescence when a 1 Likewise, the mean number offluorescence counts pfarising pM solution of PE was flowed through a focused laser beam, from a molecule in the beam is given by and they interpreted these bursts as being due to the passage uf = kfAtEf. [6] The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: PE, B-phycoerythrin. Downloaded by guest on September 29, 2021 4087 4088 Biophysics: Peck et al. Proc. Natl. Acad. Sci. USA 86 (1989) A Background Burst Emission Burst 1 r AL Ill Time B Photomultiplier Focusing Tube Laser Spectral Lens Filter Spatial /'Is Filter Flow Axis Compue FIG. 1. (A) Illustration of the concept of single-molecule fluorescence detection. When a fluorescent molecule flows through a laser beam, a burst ofphotons is generated; single-molecule events can be distinguished from the Poisson-distributed background ifthe fluorescence emission rate is higher than the background emission rate. (B) Experimental apparatus for single-molecule fluorescence detection. The 514.5-nm output from an argon-ion laser is focused to a 8-/Am-diameter spot in a capillary tube through which a sample solution is flowed. The resulting fluorescence is collected at 900 and passed through spatial and spectral filters to the detection system. When a molecule is in the beam, the probability Pf of having In other words, the observed distribution of counts is the j counts of fluorescence emission is convolution of background scattering (Pb) and fluorescence emission (Pw) occurring in an observation window ofduration PfDj) = Wfe'-f [7] At. Pw, is a modified fluorescence probability function that takes into account all possible temporal arrangements of the fluorescent molecule with respect to the observation win- When there is no fluorescent molecule in the beam, Pf(O) = dow. 1. Single molecule detection is optimized by maximizing the Fluorescence from single molecules in the beam and ratio of ff (the signal) to (nb)112 (the mean fluctuation in the scattering from solvent are two independent processes. In a background). As the light intensity increases, ff increases given time interval, the observed counts can come from any linearly until it reaches a maximal asymptotic value when possible combination of scattering and fluorescence. The the absorption rate equals the maximum emission rate. probability Ps(m, At) of observing m counts in a time interval The background nb simply increases linearly with light At is given by intensity. Thus, there should be a power at which the experiment is optimized. The most favorable illumination Ps(m, At) = >l Pb(j,At) P,(m - j,At). intensity can be experimentally determined by monitoring the Downloaded by guest on September 29, 2021 Biophysics: Peck et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4089 autocorrelation of n. The autocorrelation parameter R,(T) is amplifier/discriminator were passed to an analog moving- defined as sum circuit followed by a discriminator. The moving-sum had a response time of 280 pus, and the discriminator threshold RJ(T) = :Ej nls(j)ns(J+T) = Rb(T) + RA(T) + Rbf(T). [9] was referenced to the mean count rate (10 ms average) to correct for drift in the background count rate. Each sample R, is the sum of the autocorrelation ofnb, the autocorrelation was counted for 1000 s. ofnf, and the crosscorrelation of nb and nf. These contribu- Preparation of PE Oligomers. To 0.55 ml (3.5 mg; 25.6 ,uM) tions to R, are given by of Porphyridium cruentum PE (16) in 0.1 M sodium phos- phate/0.1 M NaCl, pH 7.40, was added 10 A.l of freshly- Rb(T) = 4j nb()fnb(i+T), [10] prepared 15 mM 3-(2-pyridyldithio)propionic acid N-hy- droxysuccinimide ester solution in methanol. After 70 min at Rf(T) = >Lj nf(j)nf(j+T), [ll] 230C, half of the reaction mixture was passed through a Sephadex G-25 (bead size, 20-80 ,um) column (0.75 x 15 cm) Rbf(T) = 2>L nf(j)nb(j+T). [12] to separate the protein derivative from excess reagent. To the other half of the reaction mixture, 10 A.l of 1 M dithiothreitol For X > 0, Rb and Rbf are independent of time because in the pH 7.40 buffer was added, and the mixture was allowed background scattering is a continuous process with Poisson- to stand at 230C for 45 min. The thiolated-PE derivative was distributed probability of occurrence. In contrast, since the separated from small molecules by gel filtration as described fluorescence emission from an individual molecule in the above.
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