Application of "in vivo cryotechnique" to detect erythrocyte oxygen saturation in frozen mouse tissues with confocal Raman cryomicroscopy

To measure oxygen saturation (SO₂) of flowing erythrocytes in blood vessels of living animals, our "in vivo cryotechnique" (IVCT) was combined with confocal Raman microscopy at low temperature (-150 °C), referred to as cryomicroscopy. We evaluated two resonance Raman (RR) shifts around 1355 and 1378 cm^-1, reflecting de-oxygenated and oxygenated hemoglobin molecular structures, respectively. Judging from the calibration analyses of quickly frozen human whole blood for the control experiment in vitro, the two RR shifts were well retained at the low temperature, and their calculated ratios mostly reflected the relative SO₂ measured with a blood-gas analyzer. In blood vessels of living mouse organs prepared with the IVCT, their RR spectral peaks were also detected at the same RR shifts obtained in human blood. In the blood vessels of living mouse small intestines, some arterioles and venules were clearly distinguishable by monitoring different peak patterns of their RR shifts. The different ratios of the RR shift-areas were calculated even in the arterial vessels. In blood vessels of mouse livers, the Raman spectra showed a lower peak shift of 1378 cm^-1 compared to that of 1355 cm^-1, indicating an SO₂ decrease in hepatic blood circulation. Thus, the new cryopreparation technique will enable us to directly analyze the in vivo SO₂ in various tissues of a whole animal body prepared with the IVCT, reflecting their living states.