We assumed that ATP could be fixed by the GA treatment during FS for the IVCT samples (Fig. 53.1c). IVCT was performed for the superficial gluteal muscles of anesthetized mice. The frozen samples were processed for the FS in acetone containing 0.2 % GA.

Skeletal muscle tissues were confirmed in the DIC mode showing structure of muscle fiber s (Fig. 53.1d). Photon images were digitally obtained with a low-light-sensitive CCD camera (Fig. 53.1e). In the skeletal muscle fibers with glutaraldehyde during FS, photon signals were abundantly detected. Numbers of photon signals between muscle fibers (M1 and M2 in Fig. 53.1d) and connective tissues (Co in Fig. 53.1e) were different. Without chemical fixative s during FS, photon signals were rarely detected. This indicates that the procedure presented here enables us to visualize ATP localization in tissue sections.

For some cryosections of skeletal muscle tissues with IVCT-FS, HE staining (Fig. 53.1f) and a diaminobenzidine (DAB) reaction (Fig. 53.1g) were performed. In capillaries, direction of blood flow was demonstrated by the helmetlike shape of erythrocytes ; the top of the helmet was thought to be the front (Fig. 53.1f). It is well known that the number of capillaries around type I muscle fiber s is higher than that around type II fibers [8] and that the sizes of the capillary-to-muscle fiber (C/F) interface differ with the tissue preparation procedure [9], although the C/F perimeter ratio was important to determine oxygen flux capacity in skeletal muscles [10]. Products of the DAB reaction were detected inside red blood cells and type I skeletal muscle fibers, which retain abundant myoglobin and mitochondrial enzymes. Imaging of the capillary network s that reflect living state s is important to evaluate the supply of oxygen to skeletal muscles. Thus, the vascular networks in skeletal muscles reflecting basic metabolism in the sedated mice were demonstrated.