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 timelapse 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 twophoton 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.

dx.doi.org/10.1364/BOE.2.001030, hdl.handle.net/1765/69171
Biomedical Optics Express
Department of Radiation Oncology

Palero, J.A, Bader, A.N, de Bruijn, H.S, van der Ploeg-van den Heuvel, A, Sterenborg, H.J.C.M, & Gerritsen, H.C. (2011). In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy. Biomedical Optics Express, 2(5), 1030–1039. doi:10.1364/BOE.2.001030