[Frontiers in Bioscience E3, 489-505, January 1, 2011]

Global analysis of autocorrelation functions and photon counting distributions

Victor V. Skakun1, Ruchira Engel2, Anatoli V. Digris1, Jan Willem Borst3, Antonie J.W.G. Visser3

1Deparment of Systems Analysis, Belarusian State University, Minsk, 220050, Belarus,2Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom, 3Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, 6703 HA Wageningen, The Netherlands


1. Abstract
2. Introduction
3. Review of theory
3.1. Fluorescence intensity distribution analysis
3.2. Photon counting histogram analysis
3.3. Fluorescence correlation spectroscopy
3.4. Correction for dynamic processes in photon counting distribution analysis
3.5. Relation between number of molecules and brightness in FCS, PCH and FIDA
3.6. Weighting of autocorrelation- and photon counting distribution functions
4. Materials and methods
4.1. Samples
4.2. Instrumentation
5. Results and discussion
5.1. General comments on global analysis
5.2. Modification of the global χ2 criterion
5.3. Monomeric and dimeric eGFPs as an experimental test system
5.4. Comparison of brightness values and diffusion times of monomeric and dimeric eGFPs
6. Conclusions
7. References


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.