Three‐dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two‐photon excitation laser scanning microscopy

Three‐dimensional maps of cellular metabolic oxidation/reduction states of rabbit cornea in situ were obtained by imaging the fluorescence of the naturally occurring reduced pyridine nucleotides (both reduced nicotinamide‐adenine dinucleotide, NADH, and reduced nicotinamide‐adenine dinucleotide phosphate, NADPH, denoted here as NAD(P)H). Autofluorescence images with submicrometre lateral resolution were obtained throughout the entire 400 μm thickness of the cornea. Two‐photon excitation scanning laser microscopy with near‐infrared excitation provided high fluorescence collection efficiency, reduced photodamage, and eliminated ultraviolet chromatic aberration, all of which have previously degraded the visualization of pyridine nucleotide fluorescence. Sharp autofluorescence images of the basal epithelium (40 μm within the cornea) show substantial subcellular detail, providing the ability to monitor autofluorescence intensity changes over time, which reflect changes in oxidative metabolism and cellular dynamics necessary for maintenance of the ocular surface. The autofluorescence was confirmed to be mostly of NAD(P)H origin by cyanide exposure, which increased the fluorescence from all cell types in the cornea by about a factor of two. Autofluorescence images of individual keratocytes in the stroma were observed only after cyanide treatment, while in the predominant extracellular collagen (> 90% of the stromal volume), fluorescence was not distinguished from the background. Observation of keratocyte metabolism demonstrates the sensitivity made available by two‐photon microscopy for future redox fluorescence imaging of cellular metabolic states. 1995 Blackwell Science Ltd