Anal. Chem. 1993, 65, 2300-2305
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Detection of Single Rhodamine 6G Molecules in Levitated Microdroplets Michael D. Barnes, Kin C. Ng,f William B. Whitten, and J. Michael Ramsey’ Analytical Chemistry Division, Oak Ridge National Laboratory, Mail Stop 6142, Oak Ridge, Tennessee 37831 -6142
Single Rhodamine 6G (R6G)molecules in levitated glycerol microdroplets have been detected with signal-to-noise ratios of >40 using CW laserinduced fluorescence. The fluorescence signal from single R6G molecules was identified by the magnitude of the fluorescence signal and by the unique time dependence of the fluorescence count rate before photobleaching. This high sensitivity allows single molecules to be counted by use of a digital detection approach offering significantly lower detection limits than those possible with conventional detection methods.
INTRODUCTION Detection of single molecules in condensed phase with high signal-to-noise ratios is important for applications which involve detection of fluorescenttags such as DNA sequencing? fluorescence immunoassay,2 or hydrology.3 In addition, observation of photophysical phenomena unique to the interaction of a radiation field with an isolated molecule, such as photon antibunching,’ depends upon having such high sensitivity. As demonstrated by the experiments of Moerner6 and Orrit: very high sensitivity at the singlemolecule level can be achieved by probing “guest” molecules in solid hosta at cryogenictemperatures. However, practical applications requiring single-molecule detection usually require measurements on liquid-phase solutions at room temperature. These demands pose new problems such as solvent-dependent quantum yield, finite fluorescence yield (the number of absorption-emission cycleswhich occur before irreversible bleaching of fluorescence), and solvent Raman scattering and fluorescence. Detection of single &phycoerythrin (a protein with 34 chromophores’) molecules in the liquid phase was demonstrated by Nguyen et al.? and Peck et d.,9 using flow cell techniques. Employing multiple-chromophore species such as 8-phycoerythrin in single-moleculedetection applications is advantageous from the standpoint of both signal magnitude and photocount statistics.10 Single R6G molecules were first t Permanent address: Department of Chemistry, California State University, Fresno, CA 93740-0070. (1) Swerdlow,H.; Chen, D. Y.; Harks, H. R.; Grey,R.; Wu, S.L.;Dovichi, N. J.; Fuller, C. Anal. Chem. 1991,63,2835. (2) Saunders, C. G.; Jett, J. H.; Martin, J. C. Clin. Chem. 1985, 31,
2020. (3) Suijlen, J.; van Leumen, W. Coastal and Estuarine, Studies; Michaelis, W. Ed.; Springer-Verlag: New York, 19w); Vol. 3. (4) Baech6, Th.;Moerner, W. E.; Orrit, M.; Talon, H. Phys. Rev. Lett. 1992,69,1516. (5) Moerner, W. E.; Kador, L. Phys. Rev. Lett. 1989,62, 2535. (6)Orrit, M.; Bernard, J. Phys. Reu. Lett. 1990, 65,2716. (7) Glazer, A. N. Mol. Cell. Biochem. 1977,18, 125. (8)Nnuven, D. C.: Keller, R. A.;. Jett.. J. H.; Martin, J. C. Anal. Chem. 1987,59; 2158. (9) Peck, K.; Stryer, L.; Glaser, A. N.; Mathies,R. A. Proc. Natl. Acad. Sci. U.S.A. 1989. 86. 4087. (10) Whitten,’W. B.; Ramsey, J. M. Appl. Spectrosc. 1992,46, 1597. 0003-2700/93/0365-2360$04.00/0
detected in the liquid phase by Shera et al.,” using a flow technique (without hydrodynamic focusing), and employed pulsed excitation and time-gated detection to observe bursts of fluorescence as molecules pass through the observation region. Indirect detection of single R6G molecules through a nonrandom autocorrelation function was also demonstrated using CW laser excitation.12 Recent extension of the timegated detection technique has shown that a mixture of two different dye molecules (with different fluorescence spectra) can be analyzed in this way using two-channel detection.13 The primary limitation of a flow technique for singlemolecule detection, however, is the trade-off between probe volume size and interaction time. For a flowing stream, the interaction time is typically limited to the time it takes the molecule to transit the probe volume defined by the laser. Obviously, interaction time can be increased by reducing the flow velocity. However, at some point, the interaction time will become limited by diffusion of the analyte molecule out of the probe volume. This problem becomes more severe as the probe volume is reduced. In addition, diffusion limits the fraction of target molecules that can be forced to transit through the optically defined probe volume. Our experimental approach to detecting single dye molecules in solution is based on using levitated microdroplets as the sample medium and employing CW laser excitation and photon-counting dete~ti0n.l~ Several advantages accrue from this approach. First, the probe volume is defined by the droplet diameter, which has been made as small as 35 fL in our experiments. Second, the analyte molecule is confined to the probe volume with no diffusive losses so that every molecule present in a droplet can be interrogated. Third, the laser-analyte interaction time can be extended arbitrarily so that all molecules remain in the interaction region until photolyzed, thus allowing the maximum number of fluorescence photons to be extracted. In addition, we have recently observed a factor of 10 increase in the spontaneous emission rate of R6G due to cavity-QED effects in 4-pm-diameter glycerol droplets.15 These effects can greatly increase the signal-to-noise ratio for single-molecule detection in two ways: through an increase in the saturated absorption rate16117 and through enhanced fluorescence yields.18 Another major potential advantage associated with microdropleta is that a significant reduction in the concentration detection limit over bulk measurement techniques can be (11) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553. (12) Soper, S. A.; Shera, E. B.; Martin, J. C.; Jett, J. H.; Hahn, J. H.; Nutter, H. L.; Keller, R. A. Anal. Chem. 1991,63,432. (13) Soper, S. A.; Davis, L. M.; Shera, E. B. J. Opt. SOC.Am. B 1992,
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9, 17f-31 . .
(14) Whitten, W. B.; Ramsey, J. M.; Arnold, S.; Bronk, B. V. Anal. Chem. 1991,63, 1027. (15) Barnes, M. D.; Whitten, W. B.; Arnold, S.; Ramsey, J. M. J. Chen. Phys. 1992,97, 7842. (16) Mathies, R. A.; Peck, K.; Stryer, L. Anal. Chem. 1990,62, 1786. (17) Nguyen, D. C.; Keller, R. A.; Trkula, M. J. Opt. SOC.Am. B 1987, 4 . 138. (18) Barnes, M. D.; Whitten, W. B.; Ramsey, J. M., submitted for publication in J . Opt. SOC. Am. B.
@ 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
achieved through "counting" of molecules or digital molecular detection. This advantage was previously recognized by Peck et al.9 and derives from dividing a sample into small segments which are analyzed sequentially. For molecules such as 8-phycoerythrin which Gaussian-like photocount statistics, extremely low concentration detection limits (c10-20M) can be achieved with modest (==lo)signal-to-noise ratios for singlemolecule detection. This technique has been recently demonstrated in the single-molecule detection of &phycoerythrin where a signal-to-noise ratio of c 5 was obtained.19 Extension of this analysis to single chromophore molecules such as R6G is nontrivial for two reasons. Obviously the first reason is the reduced fluorescence yield which will result in lower signal-to-noise ratios. The second reason is that, for a molecule such as 8-phycoerythrin, the photocount probability distribution is nearly Gaussian, whereas for single chromophore molecules, the photocount probability distribution is a decaying exponential peaked a t zero.10 Thus, the overlap between signal and noise photocount distributions is much smaller for multiple-chromophore molecules, a difference which makes "counting" analyte molecules with multiple chromophores much easier than counting molecules with only one chromophore. Therefore, much higher average signalto-noise ratios are required to efficiently count single molecules containing only one chromophore. In this paper, we report detection of single R6G molecules in micron-sized levitated glycerol droplets with signal-to-noise ratios of about 20-45 using an experimental apparatus with an optical collection efficiency approximately 1 order of magnitude greater than the system described in refs 14 and 19. Signals from individual R6G molecules were identified on the basis of both the magnitude of the signal and by the unique time dependence expected for single molecules. In addition, the distribution of measured photocounts for droplets believed to contain single R6G molecules agrees well with the distribution expected for single (one-chromophore) molecules. EXPERIMENTAL SECTION The apparatus used to measure fluorescence from levitated droplets is similar to that previously described in refs 14and 19. Briefly,dropletsare levitated ina three-electrodestructure similar to that used in ion trap mass spectrometers. An ac potential is applied to the ring electrode at 60 Hz to confine the particle, while a dc bias is applied to the two end-cap electrodes to balance againstthe gravitationalforce on the droplet. Fluorescence from the droplet was collected from the droplet with an f / l collection opticmounted in the ring electrode at an angle of 90° with respect to the CW Ar+ excitation laser. The laser was polarized in the horizontal direction, and an intensity at the droplet of -500 W/cm2was used. Light collected from the droplet was spectrally filtered using an interferencefilter centered at 575 nm with 25nm bandwidth (Omega Optical 575DF25) and focused onto a cooled photomultiplier (Hammamatsu R943-02) operated in photon-countingmode. The anode pulses were preampliied and input into a photon counter (Stanford Research SR400)with a 1-sintegration time and 2-msdwellinterval between count periods. Droplets were produced with a piezoelectric pipetmby drawing into the pipet tip about 10 pL of RGG-glycerol solutions which have been diluted in ultrapure sterile water (Carolina Biological Supply Co.) by a factor of between 20 and 50. Initially, the droplet diameter is about the same as the droplet generator tip orifice (40 pm) but rapid evaporation of water leaves a glycerol droplet with a nominal diameter of 10 pm. The droplet diameter was determined by measuring the distance between reflected and refracted glare spots21~~ from He-Ne laser illuminationusing an eyepiecereticle with rulings that correspond to 1pm. Uncertainty (19) Ng,K.C.;Whitten,W. B.; Arnold, S.;Ramsey, J. M. A w l . Chern. 1992,64,2914. (20) Arnold, S.;Folan,L. M. Rev. Sci. Zmtrurn. 1986, 57, 2250. (21) Ashkin, A.; Dziedzic, J. M. Appl. Opt. 1981, 20,1803.
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in the diameter measurement was estimated to be =lO%.a Control over the droplet diameter was achieved by varying the ratio of water and glycerol and by adjustingthe amplitude of the voltage pulse applied to the PZT in the droplet generator. The latter method tends to break the droplet into smaller "satellites" when higher voltages are used, which can result in much smaller dropletsbeing trapped. However, only a singledroplet is present in the trap during a fluorescence measurement. After careful cleaning of the droplet generator tip with semiconductor-grade methanol and sterile water, blank droplets made from a solution of sterile water and glycerol were analyzed. Blanks were prepared directly in the 1-L sterile water container. Picomolar solutions (1.8-3.6 pM) of R6G in 2% glycerol-water were prepared in the same way as the blanks. The large container volume serves to minimize diffusion and adsorption of dye molecules to the walls of the container. Fresh solutions were prepared daily, and a new working portion of the solution was drawn for each droplet.
RESULTS AND DISCUSSION Fluorescence Yield Measurements. Before attempting
to detect singlemolecules, the average number of photocounts obtained per molecule (fluorescence yield) and the photobleaching time distribution were first characterized. The average number of photons detected per molecule was measured in a separate set of experiments using relatively high (10-9-10-10 M) R6G concentrations. From a knowledge of the dye concentration and measurement of the droplet diameter, the number of molecules in the droplet was calculated. Then, the measured number of fluorescence photons (after background subtraction) was divided by the number of molecules to obtain the fluorescence yield. In addition to characterizing the sensitivity of our droplet spectrometer, these measurements also give the distribution of photobleaching times. Knowledge of this distribution is important for our single-molecule detection scheme since the signal-to-noise ratio for single-molecule detection is optimized when the integration period is approximately equal to the average photobleaching time. In contrast with flow cell methods where the transit time through the interaction region is usually much smaller than the average photobleaching time, TB,the signal-to-noiseratio for single-molecule detection in the levitated droplet technique is independent of excitation intensity provided that the counting period is approximately the same as TB. In a lowintensity regime where k,, l's (Ys,3'9, and 4's) were estimated to be 0.35, 0.37, and 0.28, respectively. The corresponding experimental fractions (0.37, 0.45, and 0.18) are in good agreement with this estimate. Part of the discrepancy between the experimental and estimated fractions is likely due to the limited sample size. However, there may be some (small) two-molecule signals likely to be accidentally included in the single-molecule photocount distribution as well as some real single-molecule signals which are excluded from the maximum likelihood analysis. Figure 6 shows the distribution of measured photocounts for the 47 droplets judged to contain single molecules from the maximum likelihood analysis. The fact that this distribution peaks at a value greater than zero is consistent with the expectation that the measured photocount distribution for single molecules should be a convolution of the signal photocount probability distribution (single-sidedexponential) and Gaussian noise distribution.10 This is illustrated by the solid overlayed curve which is the convolution of the Gaussian function shown in Figure 3 with a decaying exponential function (average number of photocounts equal to 2400). As a comparison, the distribution expected for two molecules is shown with the dashed curve. We believe that this distribution of measured photocounts lends additional support to our conclusion that we are indeed detecting single molecules since the shape of the distribution should be much different for an ensemble of droplets containing more than one molecule each. The ability to detect single R6G molecules in micrcdroplets with high signal-to-noise ratios allows us to take advantage (26) L s m n , J. L.; Uhlenbeck, G. E. Threshold Signals; McGrawHill: New York, 1950. (27) Whale", A. D.Detection of Signals in Noise; Academic Press:
San Diego. CA, 1971.
-400
ROO
200
1400
2000
2600
3200
number of photocounts Fbure 6. Hlstcgram of photocounts from single RBG molecules. The total number of samplesused in the dlsmbutbn after maxlmum Ilkdlhood analysis is 47. The solid overlaid curve represents the distribution expected lor single molecules and is the convolution 01 the Gaussian function shown in Figure 3 @ = 180. D = 200) with a single decaying exponential (average value 2400). The dashed curve shows the distribution expected for two molecules per droplet with an average number of counts per molecule of 2400.
of digitaldetection techniques whichoffer substantially lower detection limits than those possible using conventional detection methods. Digital molecular detection is essentially a technique whereby a decision is made with some degree of certainty whether a single analyte molecule is present in the probevolumeor not. The prohahilityof detecting a molecule and the probability of error can be calculated using the (singlemolecule) signal and noise distributions shown in Figures 6 and 3, respectively. If a threshold criterion for detection is used (i.e., a molecule is "detected" if the measured signal exceeds the threshold value, t ) , the probability that a single molecule can be detected is given by Pd,(t)
= j;psw(r)
dr
(3)
where t is the threshold value and p W ( r ) is the signal photocount distribution. For a threshold value of 2 0 (where ~ UN is the standard deviation of the noise distribution) t = 580, giving a probability of detecting a single molecule of 0.79 and the probability of error at the threshold of 0.21. We consider this to be a conservativeestimate since the nonzero maximum of the blank photocount distribution shown in Figure 3 can likely he attributed to the small sample size. If the noise distribution is assumed to be centered at zero (with the same standard deviation), the probability of detecting a single molecule becomes 0.85 with a probability of error at the threshold of 0.15. An additionaladvantage ofusingadigitaldetectionscheme is that the detection limit decreases exponentially with increasing signal-to-noise ratio, whereas a linear relationship exists hetween detection limit and signal-to-noise for conventional measurements. Signal-to-noise ratios can be improved in two ways in the microdroplet technique. First, the background signals can be lowered by reducing the average droplet size. Since the magnitude of the background signal goes approximately as the square of the droplet diameter. an increase in the signal-to-noise ratio of 25% can be achieved by decreasing the average droplet diameter from 10to 8 pm. Second, by reducing the diameter of the 4-5-sm range, singlemolecule fluorescence yields can be increased by as much as
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEFTEMBER 1, 1993
1order of magnitude as a result of spontaneous emission rate enhancement.18 The advantages associated with digital molecular detection derive from dividing the sample into many small segments and performing an analysis on each segment sequentially. Thus, the sensitivityadvantage of digital detection is obtained at the expense of additional measurement time. For example, to achieve concentration detection limits on the order of 10-16 M at the 90 % confidencelevel, ,105 individualmeasurements would be required (assumingan average signal-to-noiseratio of 40). Although the fluorescence can be extracted in 1or 2 s, the time required to trap the droplet and measure its diameter (=5-10 min) makes application of the levitated droplet technique to analysis of such dilute solutions impractical. We are currently developing instrumentation to perform digital chemical analysis on a stream of falling (uniformly sized) droplets so that droplets can be analyzed at kilohertz rates with signal-to-noise ratios comparable to thoee obtained using the levitated droplet approach. If digital chemical analysis is performed at fast rates with high signalto-noise ratios, the time required to make a large number of measurements will be greatly reduced, thereby making subfemtomolar concentration detection limits achievable.
CONCLUSIONS Single R6G molecules have been detected in levitated microdroplets with signal-to-noiseratios ranging between 20 and 50 using CW laser excitation and photon-counting detection. The presence of single molecules has been iden-
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tified from both the magnitude of the fluorescence signal, which agrees well with the average number of photocounts per molecule measured in separate experiments at nanomolar concentrations, and the unique time dependence of the fluorescence signal, which is significantly different from droplets containing several molecules. In addition, the photocount statistics agree well with those expected for single R6G molecules. It has previously been shown that, by employing a digital detection approach, much lower concentration detection limits may be obtained than those achievable using conventionaldetection techniques. The abilityto detect single molecules with high signal-to-noise ratios in microdroplets should provide a new approach toward analyzing ultradilute solutions.
ACKNOWLEDGMENT This research was sponsored by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract DEAC05-840R21400 with Martin Marietta Energy Systems. K.C.N. acknowledgesan appointment to the U.S. Department of Energy Faculty Research Participation Program administered by Oak Ridge Associated Universities, and M.D.B. to the ORNL postdoctoral research associate program administered by the Oak Ridge Institute for Science and Education and ORNL. RECEIVED for review March 4, 1993. Accepted June 1, 1993.
