CCD IMAGING IN CRYOSPECTROPHOTOMETRIC DETERMINATION OF MICROVASCULAR OXYHEMOGLOBIN SATURATIONS

A microspectrophotometric imaging method has been developed for locali zed measurements of intravascular oxyhemoglobin (HbO(2)) saturations i n microvessels from sections of quick-frozen tissue. HbO(2) saturation was calculated from the absorption spectrum of red blood cells measur ed at five selected wavelengths in the 520- to 570-nm range. We combin ed the use of narrow-bandwidth interference filters and a CCD camera m ounted on a microscope to obtain one gray image of the sample at each wavelength. Each pixel is a quantitative measure of transmitted light intensity from the tissue sample at that location. A linear calibratio n curve for blood frozen in vitro (humans and mice) and in vivo (mice) was obtained using a multicomponent analysis. Oxy-and deoxyhemoglobin were assumed to be the only hemoglobin components present. A constant term compensates for light loss due to scattering on red blood cells and ice crystals. The standard error in single measurements of HbO(2) saturation was 5%. The present method allows off-line analysis of the HbO(2) saturation distribution within a microvessel network and offers new possibilities for comparative morphological studies.