Three-dimensional reconstruction of living mouse liver tissues using cryotechniques with confocal laser scanning microscopy

Soluble proteins and glycogen particles are well preserved in paraffin-embedded sections prepared by in vivo cryotechnique (IVCT) and cryobiopsy followed by freeze substitution fixation. We performed confocal laser scanning microscopic analyses on the distributions of glycogen with periodic acid-Schiff (PAS) staining and serum proteins with immunostaining for mouse liver tissues. Livers of fully fed mice showed a strong fluorescence signal of PAS staining in all hepatocytes and immunofluorescence of immunoglobulin kappa light chain (Igκ) in blood vessels and bile canaliculi. However, some hepatocytes in mechanically damaged livers were PAS-negative and Igκ-immunopositive, showing extraction of glycogen particles and infiltration of serum proteins in hepatocytes. By three-dimensional (3D) reconstruction of serial optical sections, interconnecting hepatic sinusoids and bile canaliculi were detected with Igκ immunostaining between trabecular hepatocytes that were PAS stained. In PAS-stained samples under fasting conditions, interstitial structures along sinusoids were clarified in vivo by 3D reconstruction because of the lower PAS staining intensity of hepatocytes. In addition, 100-μm-thick eosin-stained slices provided 3D structural images more than 30 μm in thickness away from tissue surfaces, showing blood vessels with flowing erythrocytes and networks of bile ducts and canaliculi. IVCT and cryobiopsy with histochemical analyses enabled us to visualize native hepatocytic glycogen and 3D structures, such as vascular networks, reflecting their living states by confocal laser scanning microscopy.