Measuring Molecular Dynamics and Interactions by Förster Resonance Energy Transfer (FRET)

Halavatyi, Aliaksandr; Terjung, Stefan

doi:10.1002/9781119096948.ch4

Cited by 1 publication

(3 citation statements)

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“…through the cell cycle). 6,[8][9][10] Intensity-based FRET methods include donor intensity FRET, acceptor photobleaching (AccPb) and sensitised emission (SE-FRET). Donor intensity FRET compares the donor intensity in the presence and absence of the acceptor.…”

Section: Introductionmentioning

confidence: 99%

“…6 SE-FRET measures the acceptor intensity (illuminated by a donorexciting laser) in the presence versus absence of the donor (reviewed in Ref. [8]). However, by quantifying intensities these methods are sensitive to relative concentration and photobleaching.…”

Section: Introductionmentioning

confidence: 99%

“…FRET can therefore provide real‐time measures about two fluorescently tagged interaction partners: stoichiometry, subcellular location, proximity and the degree of interaction and its temporal dynamics (e.g. through the cell cycle) 6,8–10 …”

Section: Introductionmentioning

confidence: 99%

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Non‐fitting FLIM‐FRET facilitates analysis of protein interactions in live zebrafish embryos

Auer

Murphy

Xiao

et al. 2022

Journal of Microscopy

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Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT− FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%