Data from: 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.