IMR Press / FBE / Volume 3 / Issue 2 / DOI: 10.2741/E264

Frontiers in Bioscience-Elite (FBE) is published by IMR Press from Volume 13 Issue 2 (2021). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on imrpress.com as a courtesy and upon agreement with Frontiers in Bioscience.

Open Access Article
Global analysis of autocorrelation functions and photon counting distributions
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1 Deparment of Systems Analysis, Belarusian State University, Minsk, 220050, Belarus
2 Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
3 Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, 6703 HA Wageningen, The Netherlands

*Author to whom correspondence should be addressed,

Academic Editor: Vladana Vukojevic

Front. Biosci. (Elite Ed) 2011, 3(2), 489–505; https://doi.org/10.2741/E264
Published: 1 January 2011
Abstract

In fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis the same experimental fluorescence intensity fluctuations are used, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH yielding the molecular brightness. Both FCS and PCH give information about the molecular concentration. Here we describe a global analysis protocol that simultaneously recovers relevant and common parameters in model functions of FCS and PCH from a single fluorescence fluctuation trace. The global analysis approach is described and tested with experimental fluorescence fluctuation data of enhanced green-fluorescent protein (eGFP) and dimeric eGFP (two eGFP molecules connected by a six amino acid long linker) in aqueous buffer. Brightness values and diffusion constants are recovered with good precision elucidating novel excited-state and motional properties of both proteins.

Keywords
Fluorescence Fluctuation Spectroscopy
Fluorescence Correlation Spectroscopy
Photon Counting Histogram
Fluorescence Intensity Distribution Analysis
Global Analysis
Green Fluorescent Protein
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