A Step Forward in the Analysis of Confocal Fluorescence Spectroscopy Measurements

Fluorescence signals are commonly measured in modern microscopy experiments including confocal fluorescence spectroscopy. Typically, these signals are analyzed by well-established methods, e.g. FCS and FRAP. Although these methods provide accurate estimation of the dynamical properties of the targeted molecules, for example diffusion coefficients and photon emission rates, they require (i) long signals and (ii) high molecule concentrations which limit the scope of most recent experimental techniques, especially those probing live cells. In this presentation, we introduce a novel approach for the analysis of fluorescence signals that relaxes the requirements of existing methods and, in particular, avoids signal correlation. Specifically, our method achieves the same level of accuracy with signals that (i) are several orders shorter and (ii) produced by very low molecule concentrations. We test our method on synthetic and experimental fluorescence intensity signals from confocal microscopy. The results demonstrate the ability and accuracy of our approach to extract realistic estimates of diffusion coefficients and of other quantities of biophysical interest.