[Frontiers in Bioscience E3, 489-505, January 1, 2011] 489 Global analysis of autocorrelation functions and photon counting distributions Victor V. Skakun 1 , Ruchira Engel 2 , Anatoli V. Digris 1 , Jan Willem Borst 3 , Antonie J.W.G. Visser 3 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 TABLE OF CONTENTS 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 χ 2 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 1. 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. 2. INTRODUCTION In the last decades fluorescence correlation spectroscopy, originally introduced by Elson et al. (1-3) in the early 1970s, has become a widely used technique for studying various dynamic molecular processes (see many accounts in the 1990s (4-10)). It has found applications in measuring local concentrations, mobility coefficients, reaction rates and detection of intermolecular interactions in vitro and in vivo, excellently reviewed in this century(11- 17). The sensitivity and non-invasive nature of this technique has made it one of the important techniques for studying molecular processes in cells and thus a useful tool for biochemists, biophysicists and biologists. Fluorescence fluctuation methods are based on the detection of tiny, spontaneous fluctuations in fluorescence intensity caused due to deviations from thermal equilibrium in an open system. These fluctuations can arise e.g. due to diffusion of fluorescent molecules in and out of a well-defined observation volume generated by a focused laser beam. The intensity fluctuations can be monitored and autocorrelated over time as in fluorescence