Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.
A theory for single-molecule fluorescence detection is developed and then used to analyze data from subpicomolar solutions of B-phycoerythrin (PE). The distribution 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 autocorrelation function reveals single-molecule events and provides a criterion for optimizing experimental parameters. The transit time of fluorescent molecules through the 120-fl imaged volume was 800 microseconds. The optimal laser power (32 mW at 514.5 nm) gave an incident intensity of 1.8 x 10(23) photons.cm-2.s-1, corresponding to a mean time of 1.1 ns between absorptions. The mean incremental count rate was 1.5 per 100 microseconds for PE monomers and 3.0 for PE dimers above a background count rate of 1.0. The distribution of counts and the autocorrelation function for 200 fM monomer and 100 fM dimer demonstrate that single-molecule detection was achieved. At this concentration, the mean occupancy was 0.014 monomer molecules in the probed volume. A hard-wired version of this detection system was used to measure the concentration of PE down to 1 fM. This single-molecule counter is 3 orders of magnitude more sensitive than conventional fluorescence detection systems.
Peck
K
K
Department of Chemistry, University of California, Berkeley 94720.
Stryer
L
L
Glazer
A N
AN
Mathies
R A
RA
D007834:Lasers; D046911:Macromolecular Substances; D008433:Mathematics; D008832:Microchemistry; D010799:Phycoerythrin / Q000032:analysis*; D010860:Pigments, Biological / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
D046911:Macromolecular Substances; D010860:Pigments, Biological; D010799:Phycoerythrin