ArticleBMC methods2025
Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems.
Article in BMC methods, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.
Who cites it
2 citing papers in PubMed.
- A combined PIE-FRET and FCS assay to monitor RNA dynamics and cleavage by SARS-CoV-2 Nsp15.Scientific reports · 2026Article
- Bacterial pore-forming toxins: mechanisms and implications for host immunity.Bioscience reports · 2026Review
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
10 authors.
Funding
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 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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.