The ultrastructures of the upper surface layer of rat articular cartilage were studied with our "in vivo cryotechnique" followed by freeze-substitution method for scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Rat hip or knee articular cartilage was quickly frozen by the in vivo cryotechnique with liquid isopentane-propane cryogen (-193 degrees C), and surface areas of some frozen specimens were freeze-fractured with a scalpel in liquid nitrogen. They were freeze-substituted and freeze-dried, ion-sputtered, and then observed in SEM. Other frozen specimens were routinely freeze-substituted and embedded in epoxy resin for TEM. Many globular structures were detected in the thick upper surface layer that had not been revealed by the conventional fixation methods. Their sizes were reduced by Triton X-100 treatment, and their localization was also detected in synovial fluid, as revealed by SEM. Such globular lipid-like structures in the upper surface layer of hip or knee articular cartilage might contribute to joint lubrication.

Wistar rat kidneys were perfused with some fixatives at different pressures of 100, 150, 200 or 250 cm H₂0 via the aorta and were studied by the quick-freezing and freeze-substitution (QF-FS) or deep-etching (QF-DE) methods, in addition to the conventional immersion or perfusion fixation method. Different parts of glomerular capillary walls were selected for morphometric analyses. It was demonstrated that the widths of glomerular slit diaphragms and the spaces between foot processes were more widely dilated at higher perfusion pressures (200 and 250 cm H₂0) than those seen at both normal perfusion pressure (150 cm H₂O) and lower perfusion pressure (100 cm H₂O). On the other hand, the glomerular basement membranes were thinner at higher perfusion pressures. By the QF-FS and QF-DE methods, the foot processes showed different shapes from those revealed by the conventional preparation methods, even at the same perfusion pressure. It is concluded that the widths of glomerular slit diaphragms and glomerular basement membranes and the spaces between foot processes may be significantly changed in vivo, depending on the hemodynamics in the glomerular capillary.

The 3-dimensional ultrastructure of mouse renal glomeruli under normal haemodynamic conditions was studied by scanning electron microscopy using an in vivo cryotechnique followed by freeze-substitution, and compared with glomeruli prepared by conventional fixation methods. Mouse kidneys were frozen with a cryoknife apparatus and a liquid isopentane-propane mixture (k193 mC). Surface areas of the frozen tissues were freeze-fractured with a scalpel in liquid nitrogen. The specimens were routinely freeze-substituted, freeze-dried, ion-sputtered, and then observed in a scanning electron microscope at an accelerating voltage of 5 kV. Renal glomeruli showed good ultrastructural preservation of the surface tissues. Podocytes with interdigitating foot processes covering capillary loops exhibited smooth surface contours and their cell surfaces were arranged more tightly than those seen by the conventional fixation method. Filtration slits between foot processes were found to be narrow. The internal structure of the glomerular tuft was seen in the freeze-fracture faces. The capillary lumen with variously shaped erythrocytes was kept open in frozen glomeruli under normal blood circulation conditions. The ultrastructure of renal glomeruli, as revealed by the in vivo cryotechnique with freeze-substitution, appears to be closer to that of the living state.

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The ultrastructures of anionic sites in rat glomerular basement membranes (GBM) were studied at different perfusion pressures of 100, 150, 200 or 250 cm H₂O by a quick-freezing and deep-etching (QF-DE) method, in addition to conventional fixation methods, using polyethyleneimine (PEI) as a cationic tracer. By the QF-DE method, three-dimensional ultrastructures at each pressure were observed more clearly than those seen on conventional ultrathin sections. When the perfusion pressures were changed from low levels to higher ones, the total thickness of GBM with anionic sites became gradually thinner. Many PEI particles were observed around filaments, not only in the laminae lucidae, but also in the lamina densa. These findings indicated the existence of anionic sites in both laminae lucidae and lamina densa of GBM. The numbers of PEI particles in the lamina rara externa were counted on conventional ultrathin sections for morphometric analyses. The numbers per unit length of GBM were significantly decreased at higher perfusion pressures (200 and 250 cm H₂O) than those seen at both normal (150 cm H₂O) and lower (100 cm H₂O) pressures. It is concluded that the ultrastructures of anionic sites in the GBM may be changed in vivo, depending on the hemodynamics in the glomerular capillary.