Dual-color metal-induced and Förster resonance energy transfer for cell nanoscopy

We report a novel method, dual-color axial nanometric localization by metal-induced energy transfer, and combine it with Förster resonance energy transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells. Our approach is based on fluorescence-lifetime-imaging microscopy and allows for precise determination of the three-dimensional architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell–matrix mechanics. In addition to resolving nanometric structural details along the z-axis, we use FRET to gain precise information on the distance between actin and vinculin at focal adhesions.

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Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging

Fluorescence Microscopy ◽

10.1002/9783527671595.ch7 ◽

2013 ◽

pp. 245-291 ◽

Cited By ~ 1

Author(s):

Fred S. Wouters

Keyword(s):

Energy Transfer ◽

Fluorescence Lifetime ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Forster Resonance Energy Transfer ◽

Förster Resonance Energy Transfer ◽

Fluorescence Lifetime Imaging ◽

Lifetime Imaging ◽

Förster Resonance

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Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Scientific Reports ◽

10.1038/srep28186 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 36

Author(s):

Anca Margineanu ◽

Jia Jia Chan ◽

Douglas J. Kelly ◽

Sean C. Warren ◽

Delphine Flatters ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescence Lifetime ◽

Protein Interactions ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Forster Resonance Energy Transfer ◽

Förster Resonance Energy Transfer ◽

Fluorescence Lifetime Imaging ◽

Lifetime Imaging ◽

Förster Resonance

Abstract We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. K D values broadly agree with published biochemical measurements.

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