Observation volumes and {gamma}-factors in two-photon fluorescence fluctuation spectroscopy.

Abstract:

:Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. Although 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. We introduce a simple approach for analysis of fluorescence correlation spectroscopy 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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Nagy A,Wu J,Berland KM

doi

10.1529/biophysj.104.052779

subject

Has Abstract

pub_date

2005-09-01 00:00:00

pages

2077-90

issue

3

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(05)72852-6

journal_volume

89

pub_type

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