Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.
- 1 June 1989
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 86 (11) , 4087-4091
- https://doi.org/10.1073/pnas.86.11.4087
Abstract
A therory 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 .mu.s. The optimal laser power (32 mW at 514.5 nm) gave an incident intensity of 1.8 .times. 1023 photons .cntdot. 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 .mu.s 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.This publication has 10 references indexed in Scilit:
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