ArticleBiophysical journal2002
Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM).
Article in Biophysical journal, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 51 papers.
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Who cites it
51 citing papers in PubMed.
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- Illuminating cellular architecture and dynamics with fluorescence polarization microscopy.Journal of cell science · 2024Review
- Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.Aging cell · 2024Article
- Article
- Probing Polarity and pH Sensitivity of Carbon Dots inNanomaterials (Basel, Switzerland) · 2023Article
- Quantitative fluorescence emission anisotropy microscopy for implementing homo-fluorescence resonance energy transfer measurements in living cells.Molecular biology of the cell · 2023Article
- Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy.Journal of biomedical optics · 2023Article
- Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.Developmental cell · 2022Article
- Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy, applied to neurotransmitter imaging with GPCR-based sensors.Biomedical optics express · 2022Article
- Fluorophore spectroscopy in aqueous glycerol solution: the interactions of glycerol with the fluorophore.Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology · 2021Article
- Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer.Biophysical journal · 2021Article
- In-cell structural dynamics of an EGF receptor during ligand-induced dimer-oligomer transition.European biophysics journal : EBJ · 2020Article
- FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls.Nature methods · 2019Review
- Homotransfer of FRET Reporters for Live Cell Imaging.Biosensors · 2018Review
- Combining Graphical and Analytical Methods with Molecular Simulations To Analyze Time-Resolved FRET Measurements of Labeled Macromolecules Accurately.The journal of physical chemistry. B · 2017Article
- A Guide to Fluorescent Protein FRET Pairs.Sensors (Basel, Switzerland) · 2016Review
- Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy.Pharmaceutical research · 2016Article
- Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells.Biomedical optics express · 2015Article
- Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.International journal of molecular sciences · 2015Article
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5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and DC) between the polarized components of the fluorescence signal are recorded, leading to estimations of rotational correlation times and limiting anisotropies. Theory is developed that allows all the parameters of the hindered rotator model to be extracted from measurements carried out at a single modulation frequency. Two-dimensional image detection with a sensitive CCD camera provides wide-field imaging of dynamic depolarization with parallel interrogation of different compartments of a complex biological structure such as a cell. The concepts and technique of rFLIM are illustrated with a fluorophore-solvent (fluorescein-glycerol) system as a model for isotropic rotational dynamics and with bacteria expressing enhanced green fluorescent protein (EGFP) exhibiting depolarization due to homotransfer of electronic excitation energy (emFRET). The frequency-domain formalism was extended to cover the phenomenon of emFRET and yielded data consistent with a concentration depolarization mechanism resulting from the high intracellular concentration of EGFP. These investigations establish rFLIM as a powerful tool for cellular imaging based on rotational dynamics and molecular proximity.
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