Abstract
BACKGROUND: 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. METHODS: We describe 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. RESULTS: FRET efficiencies from both intensity- and lifetime-based analyses were consistent across systems. DNA standards reproduced expected values, RNA/DNA hybrids reported on substrate dynamics, liposome encapsulation enabled single-enzyme conformational probing, and live-cell imaging revealed transient protein-protein interactions during ribosome biogenesis. DISCUSSION: This work establishes guidelines for implementing FLIM/PIE-FRET as an accessible method to interrogate nanoscale distances, conformational dynamics, and protein-protein interactions both in vitro and in live cells. The strategies outlined here facilitate broader adoption of quantitative single-molecule time-resolved fluorescence in structural and cell biology. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s44330-025-00048-1.