Cryogenic Microspectrophotometry

Abstract

An optical method and the theoretical basis for measuring hemoglobin (Hb) and myoglobin (Mb) saturation in frozen tissue via reflected light has been applied to Mb saturation in subcellular volumes of dog gracilis muscle, a pure red muscle. This cryogenic microspectrophotometric method utilizes light intensity measurements at four quasi-monochromatic wavelengths and a modified Lambert-Beer law to calculate saturation values. Transformation from Mb saturation to tissue Po2 is based upon a Mb P₅₀ value of 5.3 Torr. Both macrocirculation and microcirculation oxygen delivery parameters are monitored as covariates. The physiologic status of Mb saturation and microcirculatory covariates at a given instant of time are obtained by rapid freezing. A 5 cm copper cube, pre-cooled to liquid nitrogen temperature, is applied to the surface of the gracilis muscle with a pressure of 0.1 kg/cm². Cooling to O°C occurs at a rate of approximately 5–10 um/ms. This rate slows as the freezing front advances into the tissue due to the decrease in temperature gradient. At 500 urn deep to the surface of the muscle, measurements indicate that −40°C is reached in < 500 ms. All measurements are made within this distance from the surface. Specimen and copper cube are maintained in contact and submerged in liquid nitrogen.