Compensation for changes in tissue light absorption in fluorometry of hypoxic perfused rat hearts

Fluorescence studies of NADH and various dyes in tissue are complicated by changes in light absorption, causing spurious results. Methods were developed to reduce and compensate for changes in light absorption in perfused rat hearts, subjected to normoxia and hypoxia. Isosbestic wavelengths were determined (i.e., absorption independent of oxygenation) in the whole ventricular wall and in the epicardium using transmitted and reflected light, respectively. Isosbestic wavelengths were found at ~385, 427, 455, 510, and 525 nm, similar in the epicardium, throughout the ventricle, in beating and arrested hearts, although the exact wavelengths varied among experiments. Furthermore, absolute light absorption was identical in the epicardium and endocardium. At nonisosbestic wavelengths, the effect of changing light absorption on fluorescence was quantified at various detection wavelengths using a reference dye. New correction methods were also developed and used to correct indo 1 fluorescence ratios for changing absorption so that results were independent of detection wavelengths. These methods can be used to greatly reduce artifacts due to changing tissue light absorption in a variety of fluorescence experiments.