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Wide-field electron microscopy. A rapid method for the study of histologic material that provides a bridge between light and electron microscopy.

Wide fields of tissue can be rapidly examined by electron microscopy by use of Formvar films for the support of ultrathin sections on slot grids. The intervention of the grid bars of conventional mesh grids is avoided, and a continuous micrograph of the specimen at scanning magnifications can be obtained. Enough material is sublimed from the section and the supporting film by deliberate exposure to the electron beam to permit one to obtain an image with good contrast. This method of examination, which takes in all about two hours, permits examination of an extensive area of tissue in relation both to its topography at low magnifications and to its ultrastructural detail, and accordingly adds to electron microscopy a dimension characteristic of the lower powers of the light microscope. It offers the histopathologist the option of using micrographs taken during the scanning survey of a tissue to detect regions that can be readily re-examined at high magnification in the same ultrathin section.

Animals

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) of biological specimens: preliminary results with a novel beam separating system.

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) utilize a parallel beam of slow-moving electrons backscattered from the specimen surface to form an image. If the electrons strike the surface an LEEM image is produced and if they are turned back just before reaching the surface an MEM image results. The applications thus far have been in surface physics. In the present study, applications of LEEM and MEM in the biological sciences are discussed. The preliminary results demonstrate the feasibility of forming images of uncoated cultured cells and cellular components using electrons in the threshold region (i.e. 0-10 V). The results also constitute a successful test of a novel beam-separating system for LEEM and MEM.

Animals

Observations of the microcirculatory bed in rat mesocecum using differential interference constrast microscopy in vivo and electron microscopy.

The microvascular bed of the rat mesocecum has been examined in vivo using differential interference (Nomarski) optics and subsequently by electron microscopy. The preferential channel, from terminal arteriole to collecting venule, has been examined. In the terminal arteriolar segment the endothelial layer is covered by a continuous layer of smooth muscle cells which, in turn, are surrounded by adventitia. In the metarteriolar segment the periendothelial cells still resemble smooth muscle cells but the tunica media is discontinuous. In the distal segment periendothelial cells are more scattered and have the appearance of pericytes. There appears to be a continuous transition of the periendothelial cell layer from terminal arteriole to distal segment. Nerve endings were seeen in both the terminal arteriolar and metarteriolar segments. During contraction smooth muscle cells, oriented circumferentially, shorten and thicken. Endothelial cells appear anchored by myoendothelial junctions. Endothelial cells have filaments which show increased banding during vasoconstriction, suggesting that such cells may contract. Capillary offshoots leave the preferential channel, usually at right angles. Smooth muscle cells are oriented to form a sphincter and there are many myoendothelial junctions at the branch point. Within a short distance the capillary branch loses its periendothelial coat.

Animals

Sephadex globules placed as landmarks in combined stereo microscopy, scanning and transmission electron microscopy.

Regions of specific interest in tissue blocks were localized in a stereo microscope and landmarked with Sephadex spheres (10-40 micron in diameter). This procedure made it possible to recognize these regions easily and quickly in the scanning electron microscope. When the tissue was later embedded in Epon the spheres facilitated orientation when sectioning. Thus it was possible with great certainty to determine the level at which ultrathin sections should be cut for transmission electron microscopy to attain an exact correspondence between scanning and transmission electron microscopic observations. The procedure is described and an example of its application is shown in a study of experimental hypertensive endothelial changes.

Angiotensin II

[Comparative endoscopic, light microscopy and scanning electron microscopy studies of the gastric mucosa in patients with chronic uremia].

Ten patients suffering from chronic uremia were selected for comparison studies of gastric mucosa. With endoscopy erosive gastritis was seen in the corpus and antrum of five patients and in the fundus of two patients. Conventional microscopy (nine patients) revealed chronic gastritis I three times in the fundus and five times in the corpus. Gastritis II was localized once in the corpus and twice in the antrum. Gastritis III was present in the antrum of four patients. Under the scanning electron microscope lesions of gastric mucosa were present in all ten cases. Single cell necrosis (A), mucosal defects involving basal membrane (B), destruction of tunica propria (C), and muscularis mucosae with bleeding (D) were seen. Mucosal lesions A and B are early stages of gastric erosions. The most severe lesions were seen in the antrum mucosa with all three methods.

Chronic Disease

Observations by immunofluorescence microscopy and electron microscopy on the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures.

The destruction of secondary mouse-embryo (ME) cells by Naegleria fowleri was studied by indirect immunofluorescence with ME-cell antiserum as a specific label to trace the fate of mammalian-cell cytoplasm. The appearance of naegleria-induced cytopathic effect in the cultures coincided with the accumulation of discrete particles containing granules of ME-cell antigen within the cytoplasm of amoebae, suggesting that the organisms ingested host-cell material. In cultures containing cytochalasin B, a non-lethal inhibitor of phagocytosis by N. fowleri trophozoites failed to acquire any granular fluorescence and were not cytopathogenic. The engulfment of mammalian-cell cytoplasm by the organisms was confirmed when thin sections of naegleria-infected ME-cell cultures were examined by electron microscopy. Amoebae were seen in the process of detaching portions of cytoplasm from whole ME cells by means of distinctive ingesting pseudopodia, and fragments of mammalian-cell cytoplasm were identified within the food vacuoles of trophozoites. There was no evidence for cytotoxic disruption of ME cells before or during engulfment of these fragments. It is concluded that N. fowleri trophozoites attack and destroy cultured ME cells by a phagocytosis-like mechanism alone, without the aid of any amoeba-associated cytotoxic or cytolytic agents. The possible significance of these findings with respect to the in-vivo pathocity of N. fowleri is discussed.

Amoeba

Scanning force microscopy and cryo-electron microscopy of tobacco mosaic virus as a test specimen.

In this study, tobacco mosaic virus (TMV) provides a resolution criterion for specimen preparation methods as well as for imaging parameters of the scanning force microscope (SFM). We present scanning force microscopic images of the virus embedded in 0.5% buffered phosphotungstic acid solution adsorbed on a freshly cleaved mica surface, and imaged under atmospheric conditions. Individual TMV particles were clearly identified with a characteristic shape of long rods of about 300 nm long and 60-70 nm in apparent width due to the geometric parameters of the tip. The structure of the virus was compared with cryo-electron microscopic data of vitrified suspensions observed to a resolution of 1.15 nm. Uncoated TMV particles were also deposited on evaporated titanium thin films and imaged by SFM.

Cryopreservation

Endothelial cell density determined by specular microscopy and scanning electron microscopy.

Human eyes were photographed with a specular microscope and later examined wit a scanning electron microscope. Corneas from patients undergoing corneal transplantation in whom we were able to obtain preoperative specular micrographs were similarly analyzed. An attempt was made to correlate the counts obtained with both microscopic techniques by determining the amount of shrinkage the cornea undergoes while being processed for SEM. All specimens were counted with a planimeter. We found that the specular microscope adequately analyzes the endothelial cell density in the central and paracentral cornea of a normal eye, but because of its small sampling area specular microscopic counts are subject to significant error when dealing with nonhomogeneous populations such as postoperative cases. We found the peripheral corneal endothelial density to be less than the central endothelial density. Furthermore, we found that we could maximize the accuracy of counting by using a variable frame in a nonhomogeneous population, counting a minimum of four photographs per specimen, analyzing different areas, and analyzing larger areas.

Adolescent

A new miniature hydrostatic pressure chamber for microscopy. Strain-free optical glass windows facilitate phase-contrast and polarized-light microscopy of living cells. Optional fixture permits simultaneous control of pressure and temperature.

This paper describes the development of a miniature, temperature-controlled, stainless steel pressure chamber which uses strain-free optical glass for windows. It is directly adaptable to standard phase-contrast and polarized-light microscopes and requires a minimum amount of equipment to generate and measure pressure. Birefringence retardation (BR) og 0.1 nm up to 3,000 psi, 0.4 nm up to 5,000 psi and 1.0 nm up to 10,000 psi can be detected over a 0.75-mm central field with two strain-free Leitz 20 times UM objectives, one used as a condenser. In phase-contrast studies a Nikon DML 40 times phase objective and Zeiss model IS long working-distance phase condenser were used, with little deterioration of image quality or contrast at pressures as high as 12,000 psi. The actual design process required a synthesis of various criteria which may be categorized under four main areas of consideration: (a) specimen physiology; (b) constraints imposed by available optical equipment and standard microscope systems; (c) mechanical strength and methods for generating pressure; and (d) optical requirements of the chamber windows. Procedures for using the chambers, as well as methods for shifting and controlling the temperature within the chamber, are included.

Atmosphere Exposure Chambers

Scanning electron microscopy of the wall of the third ventricle of the brain of Rana temporaria. III. Electron microscopy of the ventricular surface of the median eminence.

From combined scanning and transmission electron microscopic studies it is concluded (1) that all surface specializations of the median eminence of Rana temporaria are protrusions of ependymal cells; (2) that the microvillus-like structures of the median eminence are transient differentiations of the ependymal cell surface, associated with the activity of the ependymal cells.

Animals