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 Jjournal_title
Biophysical journalauthors
Nagy A,Wu J,Berland KMdoi
10.1529/biophysj.104.052779subject
Has Abstractpub_date
2005-09-01 00:00:00pages
2077-90issue
3eissn
0006-3495issn
1542-0086pii
S0006-3495(05)72852-6journal_volume
89pub_type
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