Adaptive optics for a time-resolved Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) in vivo

Förster resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have been coupled with multiphoton microscopy to image in vivo dynamics. However, the increase in optical aberrations as a function of depth significantly reduces the fluorescent signal, spatial resolution, and fluorescence lifetime accuracy. We present the development of a time-resolved FRET-FLIM imaging system with adaptive optics. We demonstrate the improvement of our adaptive optics (AO)-FRET-FLIM instrument over standard multiphoton FRET-FLIM imaging. We validate our approach using fixed cellular samples with FRET standards and in vivo with live imaging in a mouse kidney.