ArticleBiophysical journal2003
Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells.
Article in Biophysical journal, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 32 papers.
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Who cites it
32 citing papers in PubMed, 93 citations in OpenAlex.
- 4D Single-particle tracking with asynchronous read-out single-photon avalanche diode array detector.Nature communications · 2024Article
- Ultrafast fluorescence depolarisation in green fluorescence protein tandem dimers as hydrophobic environment sensitive probes.Physical chemistry chemical physics : PCCP · 2023Article
- Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy, applied to neurotransmitter imaging with GPCR-based sensors.Biomedical optics express · 2022Article
- Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer.Biophysical journal · 2021Article
- Molecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept.Frontiers in molecular biosciences · 2021Article
- Imaging FCS delineates subtle heterogeneity in plasma membranes of resting mast cells.Molecular biology of the cell · 2020Article
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- Green fluorescent protein emission obscures metabolic fluorescent lifetime imaging of NAD(P)H.Biomedical optics express · 2019Article
- Methods for Physical Characterization of Phase-Separated Bodies and Membrane-less Organelles.Journal of molecular biology · 2018Review
- The Role of Probe Photophysics in Localization-Based Superresolution Microscopy.Biophysical journal · 2017Article
- Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.Biomedical optics express · 2017Article
- Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy.The journal of physical chemistry. C, Nanomaterials and interfaces · 2017Article
- Multiphoton photochemistry of red fluorescent proteins in solution and live cells.The journal of physical chemistry. B · 2014Article
- Interaction of proteins associated with the magnetosome assembly in magnetotactic bacteria as revealed by two-hybrid two-photon excitation fluorescence lifetime imaging microscopy Förster resonance energy transfer.The journal of physical chemistry. B · 2013Article
- Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue.Proceedings of the National Academy of Sciences of the United States of America · 2011Article
- Fluorescence polarization/anisotropy in diagnostics and imaging.Chemical reviews · 2010Review
- Peripheral protein organization and its influence on lipid diffusion in biomimetic membranes.ACS chemical biology · 2010Article
- Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs.European biophysics journal : EBJ · 2010Article
- Article
- Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.Journal of structural biology · 2009Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within approximately 10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5 +/- 0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the beta-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.
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Registered trials
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