COMPENSATION FOR CHANGES IN TISSUE LIGHT-ABSORPTION IN FLUOROMETRY OFHYPOXIC PERFUSED RAT HEARTS

Fluorescence studies of NADH and various dyes in tissue are complicate d by changes in light absorption, causing spurious results. Methods we re 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 oxygenati on) in the whole ventricular wall and in the epicardium using transmit ted and reflected light, respectively. Isosbestic wavelengths were fou nd at similar to 385, 427, 455, 510, and 525 nm, similar in the epicar dium, throughout the ventricle, in beating and arrested hearts, althou gh the exact wavelengths varied among experiments. Furthermore, absolu te light absorption was identical in the epicardium and endocardium. A t nonisosbestic wavelengths, the effect of changing light absorption o n 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 re sults were independent of detection wavelengths. These methods can be used to greatly reduce artifacts due to changing tissue light absorpti on in a variety of fluorescence experiments.