Global analysis of autocorrelation functions and photon counting distributions

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.

Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells

“…Here we describe the combination of FCS and PCH in the analysis of molecular brightness and diffusion speed and its application in the analysis of dimerization of cytosolic proteins in living cells. A specific analysis technique has been developed which allows the simultaneous analysis of FCS and PCH by linking common parameters [ 26 ]. The advantage of this combined FCS and PCH global analysis method is that it enhances the advantages of PCH in estimation of brightness by adding the power of FCS in estimation of diffusion to the analysis [ 26 ].…”

Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment

“…This technique was developed to account for the fluctuations in fluorescence amplitude for molecules diffusing through an observation volume [27]. Here, we used a global PCH analysis protocol that simultaneously calculates time-dependent decay of correlation function for accurate time-indepepdent estimation of molecular brightness [28]. We beads-loaded the expression plasmids for EGFP, SGFP2 and cfSGFP2 in COS7 cells and incubated for 3 hr at 37°C, then recorded intensity fluctuation data consisting of 1–4×10 6 photons from single measurements.…”

“…Also, since the original assumption of PCH was that fluorescence intensity emitted by a molecule is constant, there is a clear effect of bin time for diffusing molecules [53]. Hence, we used a global analysis procedure that simultaneously obtains time-dependent decay of correlation function and PCH to recover relevant and common parameters [28].…”

“…The molecular brightness of each fluorescent protein was determined by finding a best-fit from globally linked datasets of a cross-correlation function and four PCH that were generated with bin time of 0.03, 0.06, 0.12 and 0.24 ms from one measuremt by using FFS data processor. A first-order correction for out-of-focus emission [51] and corrections for triplet and 3D diffusion were applied in the fitting as described [28].…”

Colorful Packages: Encapsulation of Fluorescent Proteins in Complex Coacervate Core Micelles

“…For the FCS data analysis, the FFS-data processor version 2.3 (Scientific Software Technologies Software Centre, Minsk, Belarus) was used [ 67 ]. The equation used to fit translational data, which includes triplet state, is as follows [ 68 ]: where represents the average number of fluorescent particles in the confocal volume. The exponential term describes the triplet state behavior of the molecule, in which F trip is the fraction of molecules in the triplet state and T trip is the average time a molecule resides in the triplet state.…”