SCANNING ELECTRON-MICROSCOPIC STUDY OF THE RENAL GLOMERULUS BY AN IN-VIVO CRYOTECHNIQUE COMBINED WITH FREEZE-SUBSTITUTION

The 3-dimensional ultrastructure of mouse renal glomeruli under normal haemodynamic conditions was studied by scanning electron microscopy u sing an in vivo cryotechnique followed by freeze-substitution, and com pared with glomeruli prepared by conventional fixation methods. Mouse kidneys were frozen with a cryoknife apparatus and a liquid isopentane -propane mixture (-193 degrees C). Surface areas of the frozen tissues were freeze-fractured with a scalpel in liquid nitrogen. The specimen s were routinely freeze-substituted, freeze-dried, ion-sputtered, and then observed in a scanning electron microscope at an accelerating vol tage 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 conven tional fixation method. Filtration slits between foot processes were f ound to be narrow. The internal structure of the glomerular tuft was s een in the freeze-fracture faces. The capillary lumen with variously s haped erythrocytes was kept open in frozen glomeruli under normal bloo d circulation conditions. The ultrastructure of renal glomeruli, as re vealed by the in vivo cryotechnique with freeze-substitution, appears to be closer to that of the living state.