A modification of the methods is described which makes it possible to measure pyridine nucleotide fluorescence from the brain cortex in vivo without interference from movement and hemodynamic artifacts. Movement artifacts were eliminated by the use of a window technique. Fluorescence changes due to changes in hemoglobin oxygenation have been eliminated by measuring fluorescence at an isobestic wavelength of the hemoglobin-oxyhemoglobin reaction. The interference due to changes in red blood cell concentration has been studied by simultaneous measurements of fluorescence and ultraviolet reflection. Hemodilution revealed a linear relationship between the fluorescence from the pyridine nucleotide and reflected ultraviolet light. The ratio between the light absorption changes was approximately unity under the particular optical geometry employed in this study. This method has been used to measure fluorescence changes produced by nitrogen anoxia. The technique is discussed in relation to previous methods and the effects of anoxia are compared to previous findings.
The influence of different values of arterial oxygen partial pressure (PO2) on local tissue PO2 was investigated. Tissue PO2 was measured on the surface of the beating hearts of rabbit and cat by the multiwire surface electrode as described by Kessler and Lübbers (21). In parallel experiments, local hydrogen clearance applying the H2-PH2 probe (33) was used to determine the mean blood flow per area (microflow) at the capillary level. Mean blood flow per area, v, was calculated from PH2 clearance curves obtained by local application of rectangular hydrogen pulses. The results are presented as histograms. Under steady state conditions (barbiturate narcosis), tissue PO2 ranged from 5 to 65 Torr (0.67 to 8.67 kPA) with a median of 31 Torr (4.13 kPA) in the cat heart. Mean flow per area covered values between 25 and 11 mum/s with a median of 55 mum/s. For rabbit heart muscle, the median was v = 37 mum/s and the range 28 to 46 mum/s. Hyperoxia broadened the range of tissue PO2 and shifted flow per area to lower values. Different degrees of hypoxia shifted the PO2 histogram to the left (median PO2 14 Torr and 4 Torr, respectively; [1.87 kPA and 0.53 kPA]) and the flow histogram to the right.
The construction of a microscope photometer using prefabricated elements is described. To illuminate the tissue, a Leitz Ultropac is applied. To enlarge the wavelength range, its illuminating glass lens is replaced by an Acryl glass zonal lens. Two separate light channels with separate lamps, monochromators and photomultipliers allow the measurement of fluorescence excitation and emission spectra as well as of reflection spectra. By chopping the light, light pulses and dark currents are measured 8.33 times a second. By an integration circuit the signal-to-noise ratio for small signals is improved. The instrument allows us to detect the increase of 4 ng/ml NADH (solution of pH 7.38) in an area of 0.2 mm2.
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