A reflectance spectrophotometer-surface fluorometer suitable for monitoring changes in hemoprotein spectra and fluorescence of flavins and nicotinamide nucleotides in intact tissues

A spectrophotometer-fluorometer was constructed for simultaneous measurement of reflectance spectrum changes and surface fluorescence of intact tissues and organs. The apparatus employs monochromators for dual-wavelength spectrophotometry and optical filters for fluorometry. Three light beams are time-shared by a novel type of modulator to hit the same area of the tissue to be measured. The analog circuitry was controlled by a digital logic circuitry clocked by an optically derived frequency based on the angular velocity of the rotating light modulator and produced a maximum of three sets of differences in the parameters measured. The optical design has the advantage of allowing the use of monochromators rather than filters in a multichannel apparatus. The electronics accept a wide range of light modulation frequencies without difficulties in the phasing of the detector gates, so that signal sampling can be optimized for the recording of fast and slow phenomena. The apparatus was used successfully for the simultaneous monitoring of the mitochondrial redox state and tissue oxygenation in isolated perfused rat hearts.