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Biophys. J. BioFAST: First Published July 1, 2005. doi:10.1529/biophysj.104.052779
© 2005 by the Biophysical Society.

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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Observation Volumes and Gamma Factors in Two-Photon Fluorescence Fluctuation Spectroscopy

Attila Nagy 1, Jianrong Wu 1 and Keith M. Berland 1*

1 Emory University

* To whom correspondence should be addressed. E-mail: kberland{at}physics.emory.edu.

Submitted on September 13, 2004
Revised on October 24, 2004
Accepted on 2 June 2005


  Abstract
Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. While the basic theory of fluctuation spectroscopy is well established it is not widely recognized that saturation of the fluorescence excitation can dramatically alter the size and profile of the fluorescence observation volume from which fluorescence fluctuations are measured, even at relatively modest excitation levels. A precise model for these changes is needed for accurate analysis and interpretation of fluctuation spectroscopy data. We here introduce a combined analytical and computational approach to characterize the observation volume under saturating conditions and demonstrate how the variation in the volume is important in two-photon fluorescence correlation spectroscopy (FCS). We introduce a simple approach for analysis of FCS data that can fully account for the effects of saturation, and demonstrate its success for characterizing the observed changes in both the amplitude and relaxation timescale of measured correlation curves. We also discuss how a quantitative model for the observed phenomena may be of broader importance in fluorescence fluctuation spectroscopy.

Key Words: Fluorescence correlation spectroscopy, fluctuation spectroscopy, saturation, two-photon microscopy




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Copyright © 2005 by the Biophysical Society.