Journal
CHEMPHYSCHEM
Volume 5, Issue 10, Pages 1523-1531Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cphc.200400176
Keywords
confocal microscopy; fluorescence spectroscopy; one-photon excitation; photon counting histogram
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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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