Quantitative determination of the single-molecule detection regime in fluorescence fluctuation microscopy by means of photon counting histogram analysis

The Journal of Chemical Physics
Raluca Niesner, Karl-Heinz Gericke

Abstract

Fluorescence fluctuation experiments are performed in single-molecule detection regime if the fluorescence of at most one molecule is registered at a time. Although the significance of such experiments for investigations of complex nonergodic systems like those met in the biosciences has been stressed out by many scientists, the quantitative and accurate determination of the single-molecule detection regime received rather little attention. In this work we present a method based on the photon counting histogram (PCH) analysis, which enables the determination of the average number N of molecules within the observation volume, for which only the fluorescence of individual molecules is detected at a time. Thus, the accurate design of fluorescence fluctuation experiments performed in single-molecule detection regime is possible. Demonstrative fluorescence fluctuation experiments based on two-photon excitation are performed on diluted solutions of coumarin 153, in order to verify the potential of the PCH analysis in experiments on the single-molecule detection level. If the mean number N of molecules within the excitation volume is larger than 0.048, the probability to simultaneously detect the fluorescence of two or more molecules ...Continue Reading

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Citations

Feb 7, 2013·Physical Chemistry Chemical Physics : PCCP·Markus SelmkeFrank Cichos
Jul 5, 2013·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Ouided FriaaCécile Fradin

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