17406
J. Phys. Chem. 1996, 100, 17406-17409
Photochemistry and Fluorescence Emission Dynamics of Single Molecules in Solution: B-Phycoerythrin Ming Wu,† Peter M. Goodwin, W. Patrick Ambrose, and Richard A. Keller* Chemical Science and Technology DiVision, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 ReceiVed: June 6, 1996; In Final Form: August 5, 1996X
We report the first detailed studies of the photochemistry of single molecules in aqueous solution. Photolysis of single B-phycoerythrin (B-PE) molecules, a highly fluorescent phycobiliprotein containing 34 bilin chromophores, is observed as an abrupt cessation of the fluorescence emission from individual molecules as they flow through a ∼40 pL probe volume. These measurements demonstrate that B-PE is photolyzed in a single photochemical step. The observation of abrupt photobleaching of single molecules could not be made in a bulk measurement because the signal would be averaged over many molecules to yield a continuous decay. Photon pair correlation measurements of the fluorescence detected from single B-PE molecules demonstrate that the molecule behaves as a single quantum system, not as a collection of 34 independent chromophores.
Introduction Single molecule detection (SMD) of various fluorescent species in the solid phase,1-5 in the liquid phase,6-18 and on surfaces19-22 using laser-induced fluorescence has been demonstrated by several groups over the past decade. For a review of SMD in solution see Keller et al.23 Measurements made previously on large populations can now be made at the single species level. These include fluorescence lifetime,8,11 triplet state lifetime,14,15 intersystem crossing rate,14,15 and intramolecular conformational transition rate.24 Photobleaching has been observed directly in the fluorescence emission of single molecules on surfaces20,21 and in levitated microdroplets,25 but the detailed dynamics of the process were not reported. The kinetics of a stochastic process, namely nonphotochemical persistent hole burning, were studied for single molecules in a solid.4 We report the first measurement of the kinetics of the photobleaching of individual molecules in solution. The ability to measure reaction dynamics of single molecules opens the possibility, in the future, to measure deviations from Poisson statistics as discussed by Wang and Wolynes.26 B-PE is a highly fluorescent compound belonging to a class of proteins (phycobiliproteins) found in the light harvesting structures (phycobilisomes) of red algae and cyanobacteria. B-PE is comprised of three polypeptide subunits forming a (Rβ)6γ aggregate containing a total of 34 bilin chromophores (Figure 1). The detailed crystal structure was published by Ficner et al.27 The positions and orientations of the chromophores are shown in Figure 15 of ref 27. The distances between the chromophores and the orientation factors are tabulated in this reference. The distance between the chromophores ranges from 1.9 to 11.7 nm with nearest-neighbor distances ranging from 1.9 to 3.5 nm. In aqueous solution, isolated B-PE has an intense absorption between 500 and 565 nm. The absorption cross section (σ) at 532 nm is 8.3 × 10-15 cm2,28,29 equivalent to that of ∼20 rhodamine-6G chromophores. The fluorescence quantum yield (Φf) is 0.98.28,29 B-PE fluorescence emission is peaked at 575 nm and has a lifetime † Present address: Department of Advanced Technology, Brookhaven National Laboratory, Upton, NY 11973. * To whom correspondence should be addressed. E-mail:
[email protected]. Telephone: (505) 667-3018. FAX: (505) 665-3024. X Abstract published in AdVance ACS Abstracts, October 1, 1996.
S0022-3654(96)01677-2 CCC: $12.00
Figure 1. Phycoerythrobilin, majority bilin chromophore in Bphycoerythrin.27
of ∼2.5 ns.28-30 Because of its extraordinary photophysical properties, B-PE is often used as a fluorescent label in bioanalytical applications requiring high sensitivity. B-PE was the first species to be detected at the single molecule level using laser-induced fluorescence.31,32 We report the first detailed studies of the photochemistry of single molecules in aqueous solution. Because B-PE contains 34 bilin chromophores, it might be expected that many photochemical steps are necessary to photolyze the molecule.33 This is not the case. Close examination of the time dependence of the fluorescence from single B-PE molecules reveals that they undergo photodestruction in a single photochemical step. Fluorescence from individual B-PE molecules is observed to fall abruptly to the background as a molecule crosses the laser beam. At a peak irradiance of 1.5 × 105 W cm-2 and a transit time of 6 ms, more than half of the molecules photobleach. The observation of abrubt photobleaching would not be possible in a bulk measurement because the signal would be averaged over many molecules and the fluorescence would decay continuously. Photon pair correlation measurements on fluorescence detected from individual B-PE molecules show that the 34 bilin chromophores in the molecule are strongly coupled; the molecule behaves as a single quantum system on the time scale of the fluorescence emission lifetime. Care was taken to ensure that the molecules pass through the center of the probe volume such that each molecule sees approximately the same integrated excitation irradiance, and the resulting photon burst size distribution reflects the photophysical properties of the analyte molecules. Two groups have demonstrated efficient detection of single molecules in ultradilute sample streams flowing through the center of picoliter probe © 1996 American Chemical Society
Single B-Phycoerythrin Molecules
J. Phys. Chem., Vol. 100, No. 43, 1996 17407
volumes using micropipet delivery techniques.17,34 We use micropipet delivery of B-PE to a ∼40 pL probe volume to detect single molecules with approximately 95% efficiency and to examine the photodestruction kinetics of this species from an analysis of the photon bursts. Methods Chemicals and Reagents. Water was deionized and purified on a Milli-Q Water System, Millipore (Bedford, MA). B-PE was purchased from Molecular Probes (Eugene, OR). 1× TBE buffer (Tris, boric acid, ETDA, diluted from 20× TBE) and Dulbecco’s phosphate-buffered saline (DPBS) (1×, without calcium or magnesium) were obtained from GIBCO BRL (Gaithersburg, MD). SMD Instrumentation. Our apparatus and approach have been described in detail elsewhere.35 Briefly, the second harmonic of a mode-locked Nd:YAG laser (532 nm, 70 ps pulse width, 76 MHz pulse repetition rate) was focused to a circular spot (50 µm, e-2 diameter) inside the square bore flow channel (250 × 250 µm2) of a fused silica sheath flow cuvette. At an average laser power of 6 mW, our peak irradiance (1.5 × 105 W cm-2) is below the optical saturation parameter (3 × 105 W cm-2) derived from a measurement of the fluorescence intensity as a function of laser power. Fluorescence was collected orthogonal to the flow and excitation axes with a 40×, 0.85 NA microscope objective. A slit (800 × 1200 µm2), located in the image plane of the objective with its long axis oriented parallel to the flow axis, served as a spatial filter to limit the volume from which fluorescence was collected to ∼40 pL. A band-pass filter (575 ( 15 nm) reduced the amount of Raman and Rayleigh scattered light reaching the silicon avalanche photodiode detector. A syringe pump delivered a sheath flow of ultrapure water to the flow cell at a volumetric rate of 10 µL/min to give a transit time across the laser beam of ∼6 ms. Sample was delivered to the detection volume from a 4 µm i.d. micropipet located ∼50 µm upstream of the detection volume. B-PE in 1× TBE buffer at a concentration of 2 × 10-12 M was injected into the sheath flow using gravity feed. The sample stream diameter within the detection volume was ∼20 µm. Time-correlated, single-photon counting was used to discriminate further against Raman scattered light passing through the band-pass filter. The detection efficiency for B-PE molecules delivered from the micropipet was ∼95%. Photon Pair Correlation Measurement. Approximately 0.25 mW from a continuous wave (CW) dye laser operating a 554 nm was used to excite B-PE fluorescence. A 6.3× microscope objective focused the excitation laser to a ∼7 µm, e-2 diameter spot located ∼50 µm downstream of the sample delivery capillary. Fluorescence was collected at 90° with a 40×, 0.75 NA microscope objective. A slit located in the image plane of the collection objective limited the detection volume to ∼0.5 pL. A beam splitter behind the slit directed the light to two different single photon counting avalanche photodiodes (EG&G, SPCM-200-PQ-CD-2027). Two detectors were necessary because the photodiodes have a ∼1 µs dead time, which would preclude measurement of correlations at short times with a single detector. The transmitted and reflected beams passed through separate band-pass filters (575 ( 15 nm). The outputs of the detectors were conditioned by constant fraction discriminators (CFD) and fed to the START and STOP inputs of a timeto-amplitude converter (TAC). An adjustable electronic delay, inserted between the transmitted light detector CFD output and the STOP input of the TAC, allowed the zero time delay point to be positioned near the center of the TAC measurement range (0-100 ns). Light pulses from a mode-locked dye laser
Figure 2. Photobleaching of individual B-PE molecules as they transit the probe volume. Average laser power ∼ 1 mW; beam waist ∼ 50 µm, e-2 diameter; transit time ∼ 40 ms. The bursts denoted by arrows are from molecules that photobleached.
operating at 554 nm were used to determine the time calibration and timing resolution of the apparatus (0.53 ns fwhm). B-PE, dissolved in 1× TBE buffer at a concentration of 3 × 10-8 M, was delivered electrokinetically into the sheath flow. The sheath fluid, 1× DPBS buffer, flowed at a volumetric rate of 20 µL/ min. The average number of B-PE molecules in the detection volume during the experiment was estimated to be
