Two-dimensional fluorescence lifetime correlation spectroscopy. 1. Principle.

Fluorescence correlation spectroscopy (FCS) is a unique tool for investigating microsecond molecular dynamics of complex molecules in equilibrium. However, application of FCS in the study of molecular dynamics has been limited, owing to the complexity in the extraction of physically meaningful information. In this work, we develop a new method that combines FCS and time-correlated single photon counting (TCPSC) to extract unambiguous information about equilibrium dynamics of complex molecular systems. In this method, which we name two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), we analyze the correlation of the fluorescence photon pairs, referring to the fluorescence lifetime. We first obtain the correlations of the photon pairs with respect to the excitation-emission delay times in the form of a two-dimensional (2D) map. Then, the 2D map is converted to the correlations between different species that have distinct fluorescence lifetimes using inverse Laplace transformation. This 2D FLCS is capable of visualizing the equilibration dynamics of complex molecules with microsecond time resolution at the single-molecule level. We performed a kinetic Monte Carlo simulation of a TCPSC-FCS experiment as a proof-of-principle example. The result clearly shows the validity of the proposed method and its high potential in analyzing the photon data of dynamic systems.