Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems

The structural dynamics of proteins and nucleic acids are critical for their function in many biological processes, but investigating these dynamics is often challenging with traditional techniques. Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics in a variety of systems, across many timescales. Quantitative single-molecule time-resolved fluorescence methods are uniquely positioned to investigate transient interactions and structural changes, yet application in complex biological systems remains limited by technical and analytical challenges. Combining fluorescence lifetime imaging microscopy (FLIM) with pulsed interleaved excitation Förster resonance energy transfer (PIE-FRET) offers a robust approach to overcome these barriers, enabling accurate distance measurements and dynamic studies across diverse sample types. In this study, we described practical workflows for implementing FLIM/PIE-FRET for quantitative measurements of nanoscale distances and dynamic processes in various biomolecular systems on a commercial microscope. Benchmark DNA constructs, RNA/DNA hybrids, liposome-encapsulated enzymes, and live Saccharomyces cerevisiae strains were prepared and imaged. Correction factors for FRET efficiency recovery were determined from diffusion-based experiments, and results were validated by direct comparison of intensity- and lifetime-based analyses. This data set contains raw TCSPC data (.ptu) or exported fluorescence intensity traces (.dat) for the different biomolecular samples.