Freeze cracking methods for the preparation of the scanning electron microscope specimens are described and the results obtained in the rat kidney and human spleen are demonstrated.Fixed tissue pieces immersed either in ethanol, isoamyl acetate, or in 40% dimethyl sulfoxide were quench-frozen in liquid nitrogen or in Freon-22 cooled by liquid nitrogen and cracked.Freeze cracking in ethanol and isoamyl acetate produced clean and flat fracture surfaces causing excellent visualization of the lining surfaces of the opened vessels, tubules and tissue spaces. Freeze cracking in dimethyl sulfoxide tended to cause fracture along cell surfaces and intracellular membranes.In scanning electron microscope (SEM) studies of tissues and organs, it is often desired to cut them open, not only to see the inside of some cells, but, more importantly, also to visualize some interior surfaces enclosed by cells and other elements, e.g., hepatic sinusoids, splenic sinus, urinary canalicules and capillary endothelial surfaces.

The rat kidney was perfused with saline and glutaraldehyde, treated with Murakami's tannin-osmium impregnation method, ethanol-freeze cracked and dried by the critical point method. Gold-palladium evaporated specimens were observed in a field-emission scanning electron microscope. The glomerular filtration membrane, fractured in different planes was observed with the following results: 1. Adjacent pedicles originate from different podocytes. No interpedicular bridges of apparent cytoplasmic nature could be found. 2. The basement membrane, in grazing fractures shows a horizontally layered architecture. 3. The attenuated endothelial sheet (lamina fenestrata) is divided into compartments, which we suggest should be called "areolae fenestratae", by cytoplasmic crests radiating from the nucleated portion of the endothelial cell. A crest also occurs along the cell margin, which contacts a similar crest at the margin of the adjacent cell. 4. The pores in the areolae fenestratae are variable in size (30-150 nm diameter). A knob-like projection from the apparently naked basement membrane is found in a portion of the pores. 5. Numerous microvilli may occur on the endothelium. Some of them anastomose and fuse with one another to form a net whose meshes appear identical with the endothelial pores. Domes and shelves formed of a fenestrated cytoplasmic sheet also occur above the ordinary level of the endothelial lining. A hypothesis implicating microvilli in the partial renewal of the endothelial sheet is proposed.

No abstract