Spectrally resolved fluorescence correlation spectroscopy based on global analysis

Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.