UCLA

smFRET is a powerful technique used for studying nanometer-scale dynamics of individual molecules. In solution-based smFRET, it is possible to investigate intra- and intermolecular conformations, binding and unbinding events, and conformational changes under biologically relevant conditions without ensemble averaging. However, traditional single-spot smFRET measurements in solution are inherently time-consuming.

In this study, I present a high-throughput smFRET approach that overcomes the limitations of single-spot measurements. This method utilizes a multispot confocal geometry, where excitation spots are optically coupled to two custom silicon SPAD arrays. By implementing \ac{PAX}, two-color excitation is achieved, which allows differentiation between singly- and doubly-labeled molecules, in a process called molecular sorting. By pooling data from multiple confocal spots, I demonstrate the ability of this setup to rapidly identify molecular subpopulations and accurately determine their associated FRET efficiencies.

Furthermore, this high-throughput approach enhances the temporal resolution of single-molecule FRET population characterization from minutes to seconds. When combined with microfluidics, this methodology opens doors for real-time kinetic studies and efficient molecular screening applications.

By employing this high-throughput smFRET technique, I aim to advance our understanding of single-molecule dynamics and facilitate a wide range of biophysical investigations in various fields.