Time-Shifted Fluorescence Cumulant Analysis in Fluorescent Fluctuation Spectroscopy

Abstract

Brightness analysis of fluorescence fluctuation spectroscopy (FFS) data has been developed as an analytical and experimental tool for studying protein interactions quantitatively and non-invasively in living cells. Real photon count detectors introduce dead-time and afterpulsing effects, which alter the measured photon count statistics. Careful consideration of these detector artifacts in the analysis of FFS experiments is crucial to avoid misinterpretation of FFS experimental data and is especially important for cell based studies, where large differences in concentration introduce various degrees of dead-time and afterpulsing bias. Even with careful and lengthy characterization of the detector, the correction process becomes unstable for large bias values. Brightness analysis based on the photon count distribution or its moments is strongly affected by dead-time and afterpulsing. Here, we introduce an alternative brightness analysis method called time-shifted fluorescent cumulant analysis. The new analysis leads to significantly reduced detector artifacts than the previously used conventional methods, and therefore is an attractive method for cell-based FFS studies. In this study, we demonstrate the capabilities of time-shifted fluorescence cumulant analysis and compare it with conventional brightness analysis methods. This work was supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY 0346782).