The developing inner ears of mice (CBA/CBA), ages ranging from gestational day 12 through postnatal day 21, were examined using scanning electron microscopy following the epoxy-embedding/freeze-fracture technique. This technique provides unique three-dimensional views of surface and fractured structures of the developing inner ear, thus allowing excellent preservation of the relationships between the developing sensory epithelium and the overlying membranes (i.e. the tectorial membrane and cupula) during their development. The tectorial membrane is formed of two distinct parts: the major (medial) and the minor (distal). The major portion is produced by the cells of the greater epithelial ridge prior to the formation of the minor part, which is produced largely by the primordial supporting cells of the lesser epithelial ridge. The developing tectorial membrane has two types of fibers: radial (found mainly in the major part) and slanted (found mainly in the minor part). The slanted fibers become the cover net, which fuses with the marginal band. The marginal zone of the developing tectorial membrane is completely sealed during development by the third row of Deiters' cells. The surfaces of cells that produce the tectorial membrane are characterized by numerous long microvilli which are largely lost when the tectorial membrane completely forms and separates from the supporting cells. The surface of developing auditory sensory cells is initially covered with numerous microvilli, some of which become future stereocilia. Stereocilia form stepped rows in the shape of a "W", with the tallest row located at the periphery of the cell. As the sensory cell matures, the short transitional stereocilia gradually disappear. Kinocilia on hair cells are still seen in the 14-day-old mouse (even though the organ of Corti is morphologically mature) but not in the 21-day-old mouse, indicating that complete maturation of the sensory cells in all turns is attained by 21 days of age. The mouse has upper radial tunnel fibers and basal tunnel fibers. Neural contacts of the upper radial tunnel fibers with the outer hair cells at the apical portions are frequent in the developing organ of Corti. The external sulcus cells undergo drastic changes during development, forming numerous pits that are often covered with mucus-like droplets or grape-like spherical structures of varying sizes. This phenomenon was observed only during postnatal days 6 and 14. The developing cupula starts as a thin amorphous membrane, which later becomes compact and fibrotic-like as the mass increases. By the 6th postnatal day well-developed cupular canals occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental morphology of the mouse inner ear. A scanning electron microscopic observation.

In order to observe intracellular structures by scanning electron microscopy, excess cytoplasmic matrix must be removed from the fractured surface of cells. Previously we reported an Osmium-DMSO-Osmium method devised for this purpose. This method is very effective in revealing intracellular structures, but requires osmium tetroxide for initial fixation with some consequent disadvantages. In the present study, a revised Osmium-DMSO-Osmium method is reported, in which an aldehyde mixture is used as the initial fixative instead of osmium tetroxide. As fixation is carried out by perfusion in this revised method, better preservation of fine structures is achieved than by the original method, especially in the central nervous tissue which tends to suffer from post-mortem degeneration. Moreover this method can be applied to cytochemical studies of intracellular structures with a scanning electron microscope (SEM). In this study, acid phosphatase of lysosomes is demonstrated in a coloured SEM micrograph.

A preparation method for observing intracellular structures by scanning electron microscopy

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.

Freeze cracking of scanning electron microscope specimens. A study of the kidney and spleen.

Mitochondrial structure has been examined in three dimensions using high-resolution scanning electron microscopy in cells from rat liver, retina (photoreceptors and retinal pigment epithelium), and kidney (proximal convoluted tubular cells and podocytes). Tissues were prepared by aldehyde-osmium fixation and freeze cleavage using a cryoprotectant, followed by removal of the cytosol by immersion in a dilute osmium tetroxide solution. The microscope used (Hitachi S-570) was equipped with a secondary electron detector located in the column above the specimen, situated within the objective lens. Mitochondria in all tissues examined were found to have only tubular cristae, which in some instances could be seen to span the entire diameter of the organelle. The walls of the tubular cristae, when unfractured, were in contact with the inner mitochondrial membrane; and their lumens were open to the intermembranous space. We hypothesize that in cells of many, perhaps most tissues, mitochondrial cristae are not shelf-like but are, in fact, tubes which span the mitochondrial matrix and are continuous with the inner mitochondrial membrane at both ends.

Mitochondrial structure revealed by high-resolution scanning electron microscopy.

With the development of new specimen preparation techniques and improvements in instrumental resolution, scanning electron microscopy (SEM) became an effective means of studying the three-dimensional organization of the Golgi complex. When specimens prepared by the osmium-DMSO-osmium method are observed with high-resolution SEMs, cis-most cisternae of Golgi stacks appear as sieve-like plates with many small perforations. In some cell types, larger fenestrations are also present. The trans-most cisternae showed a fenestrated or retucular pattern. At the trans-side of Golgi stacks, distinctive structures, such as a well-developed tubular plexus or a single widely extended cisterna, are observed in some types of cells. In lacrimal gland cells, Golgi stacks are linked by an irregular network of anastomosing branches extending throughout the cytoplasm. In these cells, the piled cisternae seemed to be connected to each other either directly within the stack or via cisternae of other stacks. Connections between Golgi stack and rough endoplasmic reticulum (ER) were often found in our SEM observations. In nerve cells, interconnecting tubules arise from rough ER and most often fuse with the rim of the cis-cisternae of Golgi stacks.

K. Tanaka

Papers

Freezed resin cracking method for scanning electron microscopy of biological materials.