Few-molecule spectroscopy: The three-dimensional Gaussian approximation for the confocal observation volume profile is found to be invalid in the photon counting histogram (PCH) analysis for one-photon excitation. A semiempirical approach is introduced to correct this deviation (see picture).

Abstract

The photon counting histogram (PCH) analysis is a fluorescence fluctuation method that is able to characterize the brightness and concentration of different fluorescent species present in a liquid sample. We find that the PCH model using a three-dimensional Gaussian observation volume profile is inadequate for fitting experimental data obtained from a confocal setup with one-photon excitation. We propose an improved model, which is based on the correction to the observation volume profile for the out-of-focus emission. We demonstrate that this model is able to resolve different species present under a wide range of conditions. Attention is given to how this model allows the examination of the effects of different instrumental setups on the resolvability.

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References

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Volume5, Issue10

October 18, 2004

Pages 1523-1531

Photon Counting Histogram: One-Photon Excitation

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Photon Counting Histogram: One-Photon Excitation

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