ArticleJournal of microscopy2004
Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells.
Article in Journal of microscopy, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 52 papers.
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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.
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Who cites it
52 citing papers in PubMed, 150 citations in OpenAlex.
- Fluorescence Lifetime Imaging Application to Probe GTPase Activation in Macrophage Cell Line, Using the Time-Domain FastFLIM Modality.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Advanced optical microscopy methods forBiophysical reviews · 2025Review
- Deep Learning for Fluorescence Lifetime Predictions Enables High-Throughput In Vivo Imaging.Journal of the American Chemical Society · 2025Article
- Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology.Journal of microscopy · 2025Review
- Review
- Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).Science advances · 2024Article
- Physics-Informed Masked Autoencoder for active sparse imaging.Scientific reports · 2024Article
- Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).bioRxiv : the preprint server for biology · 2024Article
- De novo design of proteins housing excitonically coupled chlorophyll special pairs.Nature chemical biology · 2024Article
- Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.International journal of molecular sciences · 2024Article
- A red-emitting carborhodamine for monitoring and measuring membrane potential.bioRxiv : the preprint server for biology · 2023Article
- Article
- YULINK regulates vascular formation in zebrafish and HUVECs.Biological research · 2023Article
- Four-dimensional multi-particle tracking in living cells based on lifetime imaging.Nanophotonics (Berlin, Germany) · 2022Article
- Integrative structural dynamics probing of the conformational heterogeneity in synaptosomal-associated protein 25.Cell reports. Physical science · 2021Article
- Direct Photon-by-Photon Analysis of Time-Resolved Pulsed Excitation Data using Bayesian Nonparametrics.Cell reports. Physical science · 2020Article
- Heterostructure andLight, science & applications · 2020Article
- Sense of Smell: Structural, Functional, Mechanistic Advancements and Challenges in Human Olfactory Research.Current neuropharmacology · 2019Article
- PucC and LhaA direct efficient assembly of the light-harvesting complexes in Rhodobacter sphaeroides.Molecular microbiology · 2016Article
- A parameter estimation method for fluorescence lifetime data.BMC research notes · 2015Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
5 authors at 2 institutions in 2 countries.
Funding
Abstract
We present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi-exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed-distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non-FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1-43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4-64, an efficient energy acceptor for FM1-43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high-resolution two-dimensional images of lifetime distributions in living neurones.
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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.