Determination of myoglobin saturation of frozen specimens using a reflecting cryospectrophotometer

This report describes a method and instrumentation for determining myoglobin (Mb) oxygen saturation in skeletal muscle. Canine gracilis is frozen in situ using a liquid N2-cooled copper block. Transverse section surfaces of frozen unstained muscle are observed at -110 degrees C using a microspectrophotometric system. The Mb saturation is determined using epi-illumination and a four-wavelength optical method. A special aperture permits illumination of a 20-microns-square area, and the radius of the catchment volume is estimated to be approximately 60 microns, with the strongest signal arising from the central region. The equibestic wavelengths used were 546.6, 570.5, and 584.1 nm. The method was validated using the nonlinear multicomponent analysis method of Lübbers. End-point (0 and 100% saturation) calibration was set using ischemic and adenosine-treated highly oxygenated muscles, respectively. The effects of hemoglobin (Hb) and metmyoglobin (metMb) signal contamination were evaluated experimentally and by computer-mixing simulations. Mb saturation determinations adjacent to large vessels are to be avoided. MetMb and capillary Hb do not interfere with the determination. The reproducibility of the method is estimated to be +/- 5%.