In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy

Nonlinear spectral imaging microscopy (NSIM) allows simultaneous morphological and spectroscopic investigation of intercellular events within living animals. In this study we used NSIM for in vivo time-lapse in-depth spectral imaging and monitoring of protein-bound and free reduced nicotinamide adenine dinucleotide (NADH) in mouse keratinocytes following total acute ischemia for 3.3 h at ~3 min time intervals. The high spectral resolution of NSIM images allows discrimination between the two-photon excited fluorescence emission of protein-bound and free NAD(P)H by applying linear spectral unmixing to the spectral image data. Results reveal the difference in the dynamic response between protein-bound and free NAD(P)H to ischemia-induced hypoxia/anoxia. Our results demonstrate the capability of nonlinear spectral imaging microscopy in unraveling dynamic cellular metabolic events within living animals for long periods of time.