ArticleScientific reports2020
QuanTI-FRET: a framework for quantitative FRET measurements in living cells.
Article in Scientific reports, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed.
- Protocol for multiplexed quantification of fluorescent biosensor FRET efficiency using barcoded cells.STAR protocols · 2026Article
- Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding.iScience · 2025Article
- Direct Quantification of Protein-Protein Interactions in Living Bacterial Cells.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Article
- Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology.Journal of microscopy · 2025Review
- Article
- Fiber photometry in neuroscience research: principles, applications, and future directions.Pharmacological reports : PR · 2024Review
- Detection of fluorescent protein mechanical switching in cellulo.Cell reports methods · 2024Article
- In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET.Biophysical reports · 2023Article
- Article
- Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy improves accuracy for samples with spatially varied autofluorescence to signal ratio.Scientific reports · 2023Article
- Detecting Förster resonance energy transfer in living cells by conventional and spectral flow cytometry.Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2022Article
- Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of alpha-synuclein and tau.Nature communications · 2022Article
- Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.The Journal of chemical physics · 2022Article
- Inside a Shell-Organometallic Catalysis Inside Encapsulin Nanoreactors.Angewandte Chemie (International ed. in English) · 2021Article
- Simultaneous readout of multiple FRET pairs using photochromism.Nature communications · 2021Article
- FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.eLife · 2021Review
- Unraveling protein's structural dynamics: from configurational dynamics to ensemble switching guides functional mesoscale assemblies.Current opinion in structural biology · 2021Review
- UNMIX-ME: spectral and lifetime fluorescence unmixing via deep learning.Biomedical optics express · 2020Article
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Authors and funding
8 authors.
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No grant is acknowledged in the PubMed record.
Abstract
Förster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. This sensitivity is regularly achieved in single-molecule experiments in vitro but is still challenging in biological materials. Despite many efforts, quantitative FRET in living samples is either restricted to specific instruments or limited by the complexity of the required analysis. With the recent development and expanding utilization of FRET-based biosensors, it becomes essential to allow biologists to produce quantitative results that can directly be compared. Here, we present a new calibration and analysis method allowing for quantitative FRET imaging in living cells with a simple fluorescence microscope. Aside from the spectral crosstalk corrections, two additional correction factors were defined from photophysical equations, describing the relative differences in excitation and detection efficiencies. The calibration is achieved in a single step, which renders the Quantitative Three-Image FRET (QuanTI-FRET) method extremely robust. The only requirement is a sample of known stoichiometry donor:acceptor, which is naturally the case for intramolecular FRET constructs. We show that QuanTI-FRET gives absolute FRET values, independent of the instrument or the expression level. Through the calculation of the stoichiometry, we assess the quality of the data thus making QuanTI-FRET usable confidently by non-specialists.
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