FRET imaging

Jares‐Erijman, Elizabeth A.; Jovin, Thomas M.

doi:10.1038/nbt896

Cited by 1,781 publications

(1,372 citation statements)

References 83 publications

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“…FLIM is frequently applied to imaging bimolecular interactions such as Förster resonance energy transfer (FRET), which reports on the distance between a donor and acceptor molecule, e.g. [34], [35]. FLIM-FRET is attractive compared to intensity-based imaging techniques because the fluorescence decay profiles depend on the excited state reactions but not on fluorophore concentration or optical path length [7].…”

Section: Flim Microscopy Applied To Investigate Fluorophore Environmentmentioning

confidence: 99%

Time-domain fluorescence lifetime imaging applied to biological tissue

Elson

Requejo-Isidro

Munro

et al. 2004

Photochem Photobiol Sci

177

135

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Fluorescence lifetime imaging (FLIM) is a functional imaging methodology that can provide information, not only concerning the localisation of specific fluorophores, but also about the local fluorophore environment. It may be implemented in scanning confocal or multi-photon microscopes, or in wide-field microscopes and endoscopes. When applied to tissue autofluorescence, it reveals intrinsic excellent contrast between different types and states of tissue. This article aims to review our recent progress in developing time-domain FLIM technology for microscopy and endoscopy and applying it to biological tissue.

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Section: Flim Microscopy Applied To Investigate Fluorophore Environmentmentioning

confidence: 99%

Time-domain fluorescence lifetime imaging applied to biological tissue

Elson

Requejo-Isidro

Munro

et al. 2004

Photochem Photobiol Sci

177

135

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“…Since FRET only occurs between molecules in close proximity, it can be used as a spectroscopic method to investigate interactions of and conformational changes in biological macromolecules. 5,6 Since recently, improved opto-electronic technology in cell biology 7,8 allows imaging of biological processes in living cells with high speed and accuracy using genetically encoded fluorescence sensors.…”

Section: ■ Introductionmentioning

confidence: 99%

Disentangling Picosecond Events That Complicate the Quantitative Use of the Calcium Sensor YC3.60

Laptenok

Stokkum

Borst³

et al. 2012

J. Phys. Chem. B

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Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Forster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyanfluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time-and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca 2+ concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.

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“…For example, assembly of protein complexes during signal transduction processes increases the speed of enzymatic reactions, ensures the specificity of signaling and targets signaling molecules to proper intracellular compartments. During recent years, fluorescence resonance energy transfer (FRET) has become a key method for the analysis of proteinprotein interactions during signal transduction in living cells [1][2][3] . FRET studies using proteins tagged with mutant derivatives of the green fluorescent protein (GFP) have demonstrated the formation of complexes of signaling proteins in various intracellular compartments [4][5][6][7] .…”

mentioning

confidence: 99%

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

2004

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Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer . This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.Many cellular processes are governed by multi-component molecular machineries that rely on dynamic and highly coordinated protein-protein interactions. For example, assembly of protein complexes during signal transduction processes increases the speed of enzymatic reactions, ensures the specificity of signaling and targets signaling molecules to proper intracellular compartments. During recent years, fluorescence resonance energy transfer (FRET) has become a key method for the analysis of proteinprotein interactions during signal transduction in living cells [1][2][3] . FRET studies using proteins tagged with mutant derivatives of the green fluorescent protein (GFP) have demonstrated the formation of complexes of signaling proteins in various intracellular compartments 4-7 . FRET-based genetically encoded biosensors for second messengers, protein phosphorylation and activity of small GTPases have provided insights into the spatial and temporal regulation of signaling processes [8][9][10][11][12] .The combination of enhanced cyan (CFP) and yellow (YFP) fluorescent proteins has proven to be most effective in many FRET studies. Cloning of Anthozoa fluorescent proteins, such as red DsRed 13,14 and far-red HcRed 15 , has expanded the in vivo applications of FRET, but a major limitation of Anthozoa proteins for FRET applications is their obligate oligomerization 16 . Recent generation of a monomerized mutant of DsRed, monomeric red fluorescent protein 1 (mRFP) 17 , provides the opportunity to generate functional monomeric fusion proteins and to use mRFP as a FRET acceptor with proteins fused to GFP or its mutants.Various methods of FRET measurements have been used to visualize protein-protein interactions 18 . The general limitation of these methods is that they only permit the analysis of interactions between two proteins. To analyze multi-component signaling complexes, a method to measure interactions betwe...

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FRET imaging

Cited by 1,781 publications

References 83 publications

Time-domain fluorescence lifetime imaging applied to biological tissue

Time-domain fluorescence lifetime imaging applied to biological tissue

Disentangling Picosecond Events That Complicate the Quantitative Use of the Calcium Sensor YC3.60

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

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