Photon Counting Histogram: One-Photon Excitation

• Published in: Chemphyschem Vol. 5; no. 10; pp. 1523 - 1531
• Main Authors: Huang, Bo, Perroud, Thomas D., Zare, Richard N.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 18.10.2004; WILEY‐VCH Verlag; Wiley
• Subjects: Atomic and molecular physics; confocal microscopy; Exact sciences and technology; Fluorescence and phosphorescence spectra; Fluorescence and phosphorescence; radiationless transitions, quenching (intersystem crossing, internal conversion); fluorescence spectroscopy; Molecular properties and interactions with photons; one-photon excitation; photon counting histogram; Physics; Fluorescence spectroscopy; Chemical solution; Confocal microscopy; photon counting histogram; Photon counting; one-photon excitation
• ISSN: 1439-4235; 1439-7641
• DOI: 10.1002/cphc.200400176

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. 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).