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Proceedings Paper

Saturation effects in fluorescence correlation spectroscopy
Author(s): Lloyd M. Davis; Guoqing Shen; David A. Ball
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Paper Abstract

Fluorescence correlation spectroscopy (FCS) could provide a more useful tool for intracellular studies and biological sample characterization if measurement times could be reduced. While an increase in laser power can enable an autocorrelation function (ACF) with adequate signal-to-noise to be acquired within a shorter measurement time, excitation saturation then leads to distortion of the ACF and systematic errors in the measurement results. An empirical method for achieving reduced systematic errors by employing a fitting function with an additional adjustable parameter has been previously introduced for two-photon FCS. Here we provide a unified physical explanation of excitation saturation effects for the three cases of continuous-wave, pulsed one-photon excitation, and two-photon excitation FCS. When the time between laser pulses is longer than the fluorescence lifetime, the signal rate at which excitation saturation occurs is lower for pulsed excitation than for cw excitation, and due to the disparate timescales of the photophysical processes following excitation, it is lower still for two-photon excitation. We use a single-molecule description of FCS to obtain improved analytical ACF fitting functions for the three cases. The fitting functions more accurately account for saturation effects than those previously employed without the need for an additional empirical parameter. Use of these fitting functions removes systematic errors and enables measurements to be acquired more quickly by use of higher laser powers. Increase of background, triplet photophysics, and the cases of scanning FCS and fluorescence cross-correlation spectroscopy are also discussed. Experimental results acquired with a custom built apparatus are presented.

Paper Details

Date Published: 30 March 2005
PDF: 10 pages
Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); doi: 10.1117/12.591099
Show Author Affiliations
Lloyd M. Davis, Univ. of Tennessee (United States)
Guoqing Shen, Univ. of Tennessee (United States)
David A. Ball, Univ. of Tennessee (United States)

Published in SPIE Proceedings Vol. 5700:
Multiphoton Microscopy in the Biomedical Sciences V
Ammasi Periasamy; Peter T. C. So, Editor(s)

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