Scanning electron microscopic study of the renal glomerulus by an in vivo cryotechnique combined with freeze-substitution

Abstract

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 (−193°C). 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.