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Chemphyschem. 2004 Oct 18;5(10):1523-31.

Photon counting histogram: one-photon excitation.

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  • 1Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.


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 imoroved 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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