Abstract
Microscopy methods used to measure Förster resonance energy transfer (FRET) between fluorescently labeled proteins can provide information on protein interactions in cells. However, these methods are diffraction-limited, thus do not enable the resolution of the nanodomains in which such interactions occur in cells. To overcome this limitation, we assess FRET with an imaging system combining fluorescence lifetime imaging microscopy with stimulated emission depletion, termed fluorescence lifetime imaging nanoscopy (FLIN). The resulting FRET-FLIN approach utilizes immunolabeling of proteins in fixed cultured neurons. We demonstrate the capacity to discriminate nanoclusters of synaptic proteins exhibiting variable degrees of interactions with labeled binding partners inside dendritic spines of hippocampal neurons. This method enables the investigation of FRET within nanodomains of cells, approaching the scale of molecular signaling.
Highlights
To understand cell signaling at the molecular level, the capacity to monitor and resolve molecular interactions at their scale is mandatory
We evaluated the benefits of performing Förster resonance energy transfer (FRET) with fluorescence lifetime imaging nanoscopy (FRETFLIN) over FRET-Fluorescence lifetime imaging microscopy (FLIM), using simulation, and compared different fluorescence lifetime analysis approaches
Tardif et al.: Fluorescence lifetime imaging nanoscopy for measuring Förster resonance energy transfer in cellular nanodomains surrounding the center of the point spread function
Summary
To understand cell signaling at the molecular level, the capacity to monitor and resolve molecular interactions at their scale is mandatory. Förster resonance energy transfer (FRET), a physical nonradiative process that occurs between an excited fluorophore (donor) and another fluorophore (acceptor), can be measured to evaluate molecular proximity. Combined to molecular spectroscopy or optical imaging techniques, it allows accurate distance measurements between interacting molecules at the nanoscale (
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