Maximum Entropy Method-Based Acceptor Triplet-State Fluorescence Correlation Spectroscopy Analysis for Determination of Donor–Acceptor Distance Distribution: Theory and Simulation

A maximum entropy method-based acceptor triplet-state fluorescence correlation spectroscopy (tsFCS) analysis, i.e., tsFCS fluorescence resonance energy transfer (tsFCS-FRET), was proposed to resolve the donor-acceptor distance (R) distribution of a FRET system with multiple distances. An R-dependent acceptor triplet-state weight distribution function was introduced into the excited tsFCS model with a fixed R, and the weight distribution maximized the value of the Shannon entropy. tsFCS-FRET analysis showed consistent distributions with the actual pre-input for both unimodal distribution and two-species systems and a distribution with three peaks close to the pre-inputted values for three-species system at higher donor laser power. Collectively, tsFCS-FRET provides a powerful tool for resolving the R distribution, in turn, to the FRET efficiency (E) distribution, of a FRET system containing multiple E species.