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Journal of Biomedical Optics

Characterizing observation volumes and the role of excitation saturation in one-photon fluorescence fluctuation spectroscopy
Author(s): Attila Nagy; Jianrong Wu; Keith M. Berland
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Paper Abstract

Fluorescence correlation spectroscopy (FCS) and related distribution analysis techniques have become extremely important and widely used research tools for analyzing the dynamics, kinetics, interactions, and mobility of biomolecules. However, it is not widely recognized that photophysical dynamics can dramatically influence the calibration of fluctuation spectroscopy instrumentation. While the basic theories for fluctuation spectroscopy methods are well established, there have not been quantitative models to characterize the photophysical-induced variations observed in measured fluctuation spectroscopy data under varied excitation conditions. We introduce quantitative models to characterize how the fluorescence observation volumes in one-photon confocal microscopy are modified by excitation saturation as well as corresponding models for the effect of the volume changes in FCS. We introduce a simple curve fitting procedure to model the role of saturation in FCS measurements and demonstrate its accuracy in fitting measured correlation curves over a wide range of excitation conditions.

Paper Details

Date Published: 1 July 2005
PDF: 9 pages
J. Biomed. Opt. 10(4) 044015 doi: 10.1117/1.1991860
Published in: Journal of Biomedical Optics Volume 10, Issue 4
Show Author Affiliations
Attila Nagy, Research Institute for Solid-state Physics and Optics (Hungary)
Jianrong Wu, Emory Univ. (United States)
Keith M. Berland, Emory Univ. (United States)

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