Two-dimensional analysis of the redox state of the rat cerebral cortex in vivo by NADH fluorescence photography

A photographic method for measuring two-dimensional changes in NADH fluorescence and hemoglobin distributions in the rat cerebral cortex in vivo has been developed. Intracellular NADH was excited by UV light peaking at 360 nm and the emission was observed through a window with the maximum transmission at 450 nm. The fluorescence photographs (360 → 450 nm) required 20-25 sec exposures at the aperture opening of f/5.6 and the reflectance photographs (360 → 360 nm) 10 sec exposures at f/32. The digitization of photographic images was achieved either by a PDP-8-controlled microdensitometer coupled to an A/D converter or by a combination of a manually operated microdensitometer and a computer-controlled digitizer. In the latter case, a photographic negative was scanned with a Joyce-Loebl microdensitometer in parallel lines 170 μm apart, and the densitometric tracings were digitized with a PDP-8-controlled TV digitizer. The digital data were processed by DEC PDP-10 computer and the results were displayed in 3-dimensional surfaces. Nitrogen anoxia caused increases in fluorescence at 450 nm ranging from 10 to 75% of the normoxic fluorescence intensities (after correcting for the logarithmic characteristics of the photographic films) and decreases in reflectance intensities in the range of 10-30%. The spatial resolution of the present technique is limited to approximately 30 μm × 30 μm on the cortical surface and the time resolution to 10-25 sec. The optical properties of the cerebral cortex in vivo appear to be controlled primarily by blood vessel patterns and hemodynamic factors and secondarily by the redox state of the tissue. Evidence for a heterogeneous redox response of the cerebral cortex toward N₂ anoxia was obtained.