Two-Photon FRET/FLIM Imaging of Cerebral Neurons.

Two-photon FRET (Förster resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) enable the detection of FRET changes of fluorescence reporters in deep brain tissues, which provide a valuable approach for monitoring target molecular dynamics and functions. Here, we describe two-photon FRET and FLIM imaging techniques that allow us to visualize endogenous and optogenetically induced cAMP dynamics in living neurons with genetically engineered FRET-based cAMP reporters.