The small intestines with IVCT [9] were analyzed with confocal Raman cryomicroscopy (Fig. 44.1b). Blood vessel s were exposed by freeze-fractured with a cooled scalpel ((ii) in Fig. 44.1b). Their intestinal mesenteries and serous membranes contain many typical small arteries and veins. The shift peaks around 1355 and 1378 cm⁻¹ of the frozen intestinal tissues completely matched the RR shifts of HWB, indicating a common characteristic in all animal hemoglobin molecules [10].

Under a light microscope, some branches of the small arteries and arterioles were identified as bright red color images (Fig. 44.1c), and Raman shifts in the blood vessel s were analyzed with confocal Raman cryomicroscopy . In some arterial vessels, depending on the peripheral distance from one point of an artery, area ratios of the two shift peaks changed (Fig. 44.1d); the 1378 cm⁻¹ Raman shift peaks decreased with distance compared to the 1355 cm⁻¹ ones. Thus, it was possible to obtain Raman spectra of frozen tissue samples at low temperature , indicating the SO₂ of flowing erythrocyte s .

The liver tissues frozen with IVCT were also examined to verify the RR spectra in blood vessel s at low temperature . With confocal Raman cryomicroscopy , two shifts around 1355 and 1378 cm⁻¹ were detected in the blood vessels of liver tissues, as prepared by IVCT, under the normal circulation. The Raman spectra were also compared in two neighboring blood vessels. Judging from the relative area ratios of 1355 and 1378 cm⁻¹ RR shifts, the SO₂ in the hepatic blood vessels was much lower than that in the mesenteric arterioles . However, in blood vessels of the hypoxic areas after blood circulation stopped, the peak of the 1378 cm⁻¹ RR shift was completely lost in both blood vessels, indicating much lower levels of SO₂ in the flowing erythrocyte s under hypoxia .