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23 February 2006 Wave optics analysis of observation volumes in two-photon fluorescence fluctuation spectroscopy
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Abstract
Information recovery in fluorescence fluctuation spectroscopy requires accurate models both for the physical dynamics observed and for the effective size and shape of the sample region from which fluorescence signals are measured. In both one- and two-photon fluctuation spectroscopy, the so called observation volume is assumed to be well approximated by a three dimensional Gaussian (3DG) function. Here, we present wave optics computations that provide an accurate representation of the true profile for the fluorescence measurement with two-photon excitation. Fluorescence correlation spectroscopy (FCS) curves are computed for these true profiles for a variety of optical configurations, and we demonstrate that under most illumination conditions the 3DG based FCS models do provide reasonable approximations to the measured FCS curves.
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Jianrong Wu and Keith Berland "Wave optics analysis of observation volumes in two-photon fluorescence fluctuation spectroscopy", Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 60890M (23 February 2006); https://doi.org/10.1117/12.660870
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