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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.

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