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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.

Fluorescence lifetime imaging nanoscopy for measuring Förster resonance energy transfer in cellular nanodomains