FoCuS-point: software for STED fluorescence correlation and time-gated single photon counting

Motivation: Fluorescence Correlation Spectroscopy (FCS) is a popular tool for measuring molecular mobility and how mobility relates to molecular interaction dynamics and bioactivity in living cells. The FCS technique has been significantly advanced by its combination with super-resolution STED microscopy (STED-FCS). Specifically, the use of gated detection has shown great potential for enhancing STED-FCS, but has also created a demand for software which is efficient and also implements the latest algorithms. Prior to this study, no open software has been available which would allow practical time-gating and correlation of point data derived from STED-FCS experiments. Results: The product of this study is a piece of stand-alone software called FoCuS-point. FoCuS-point utilizes advanced time-correlated single-photon counting (TCSPC) correlation algorithms along with time-gated filtering and innovative data visualization. The software has been designed to be highly user-friendly and is tailored to handle batches of data with tools designed to process files in bulk. FoCuS-point also includes advanced fitting algorithms which allow the parameters of the correlation curves and thus the kinetics of diffusion to be established quickly and efficiently.