Cryobiopsy was developed for clinical and repeated collection as substitute for our “in vivo cryotechnique .” To examine that the cryobiopsy was time-dependently repeatable, one piece from an area of liver tissues was obtained by cryobiopsy (Fig. 51.2a-i), and another area was cryobiopsied for 6 h (Fig. 51.2c-ii). Temporary bleeding from the tissue area pinched with the cryoforceps was quickly stopped by covering it with instant glue (Fig. 51.2b). During the continuous experiments, all the mice were warmed on an electrically heated plate to maintain their body temperature. In the liver tissues of an almost fully fed mouse, PAS staining was observed in almost all hepatocytes (Fig. 51.2a), with a slightly lower intensity in some hepatocytes around portal vein s . After 6-h fasting, the PAS-staining intensity of hepatocytes had clearly decreased around the portal tracts (Fig. 51.2b), but other hepatocytes far from the portal veins in zone I were still PAS positive (arrowheads in Fig. 51.2b). Thus, the time-dependent changes in glycogen distribution can be clearly evaluated in a single mouse liver by cryobiopsy.

To examine the effects of blood circulation on glycogen consumption, temporary stopping and reflowing of blood circulation were produced by binding with a circular clip and removing it (Fig. 51.3a). To prevent drying of the liver surface tissues, abdominal skin was quickly sutured, and a cotton sheet-absorbing physiological saline was placed on the mouse abdomen throughout the experiment. Temporary hypoxia of some parts of the clipped liver tissues had already been confirmed by measuring the oxygen saturation of similar liver tissues with confocal Raman cryomicroscopy , as previously reported [17]. After stopping blood circulation for 30 min and reflowing for 1 h, cryobiopsy of the restricted areas was performed on the inside (Fig. 51.3b-I) or outside (Fig. 51.3b-II) of the clipped liver tissues. In the liver tissues inside the clipped areas, the PAS -staining intensity was markedly decreased in certain areas around the portal vein s of zone I and also the central vein s (zone III) (Fig. 51.3c). Even in liver tissues outside the clipped areas (Fig. 51.3c, d), the PAS-staining intensity heterogeneously decreased as a mosaic pattern in all zonal areas. Thus, the PAS staining of liver tissues by cryobiopsy clearly revealed zonal difference s in glycogen storage after reflowing of blood circulation in living mice and probably reflects zonal metabolic functions in vivo.