FCS is an incredibly versatile technique convenient for probing the time-dependent properties of biological systems. In particular, FCS can be used to readout quantities such as concentration, translational diffusion coefficients, and hydrodynamic radii as well as provide information regarding the rotational dynamics and reaction kinetics within a system when an appropriate model is known. However, routine FCS alone is limited in its capability to capture all pertinent reaction parameters associated with complex biochemical reactions, including two-state binding reactions or reactions in which the interacting components have different brightness.
To overcome this limitation, our goal is to advance the higher-order FCS method that was originally proposed by Palmer and Thompson in the late 1980s and refers to the computation and analysis of third- or higher-order correlation functions in addition to the second-order functions obtained from routine analysis. Recently, the group developed a sub-binning approach that provides a time resolution on the order of microseconds with a standard two-detector FCS setup and alleviates detector artifacts such as detector dead time and shot-noise, which are typically amplified in higher-order correlation analysis. As a proof-of-concept, the higher-order FCS method with sub-binning was applied to a model DNA hairpin system, from which a three-state folding mechanism.
Example higher-order correlation functions with corresponding rate constants and brightness values describing the two- and three-state mechanism of DNA hairpin folding.