Characterizing observation volumes and the role of excitation saturation in one-photon fluorescence fluctuation spectroscopy

A. Nagy, Jianrong Wu, Keith M. Berland

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish
Article number044015
JournalJournal of Biomedical Optics
Volume10
Issue number4
DOIs
Publication statusPublished - Jul 2005

Fingerprint

Fluorescence Spectrometry
Photons
Spectrum Analysis
Fluorescence
Observation
Spectroscopy
saturation
fluorescence
photons
spectroscopy
excitation
Confocal Microscopy
Calibration
Confocal microscopy
curve fitting
Curve fitting
Biomolecules
Research
microscopy
Kinetics

Keywords

  • Fluorescence correlation spectroscopy
  • Observation volume
  • Saturation

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biomaterials
  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging
  • Radiological and Ultrasound Technology
  • Clinical Biochemistry

Cite this

Characterizing observation volumes and the role of excitation saturation in one-photon fluorescence fluctuation spectroscopy. / Nagy, A.; Wu, Jianrong; Berland, Keith M.

In: Journal of Biomedical Optics, Vol. 10, No. 4, 044015, 07.2005.

Research output: Contribution to journalArticle

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