Fine structural cytology of the adenohypophysis in rat and man

The present review deals with the use of electron microscopy in the identification of pituitary cell types as well as the assessment of their functional state, in rat and man. Application of immunoelectron microscopy, especially immunogold techniques, utilizing multiple labeling in establishing differentiation and hormone content of cell types, is emphasized. Recent evidence of plurihormonality in various pituitary cell types indicates that the once axiomatic one cell-one hormone theory is untenable and that the present perception of pituitary cell types and their function requires modification. Detection of hormonal and nonhormonal substances in pituitary cell types, not associated with their known endocrine function, suggests that hypophysial cells may have yet unknown roles, possibly in the realm of paracrine and autocrine regulation.     

Preparation of carbon films from evaporated fibers and rods at fore-vacuum pressure

Fore-vacuum (pressure ≥ 3 Pa) evaporation of carbon fibers and rods to thin films has been tested and the resulting thicknesses recorded. A modified sputter coater was used as a vacuum evaporator. The quality of the carbon films was evaluated by bright-field and dark-field electron microscopy. Although high-vacuum-evaporated carbon films are superior in quality, low-vacuum-evaporated carbon films were found fully acceptable for routine work in bright field, for both TEM and SEM purposes. Apart from being time-saving, the method presented has the obvious advantage, in SEM preparations, that carbon coating and metal sputtering can be carried out in the same unit without breaking the vacuum.     

Phase differentiation via combined EBSD and XEDS

Electron backscatter diffraction (EBSD) and orientation imaging microscopy have become established techniques for analysing the crystallographic microstructure of single and multiphase materials. In certain instances, however, it can be difficult and/or time intensive to differentiate phases within a material by crystallography alone. Traditionally a list of candidate phases is specified prior to data collection. The crystallographic information extracted from the diffraction patterns is then compared with the crystallographic information from these candidate phases, and a best‐fit match is determined. Problems may arise when two phases have similar crystal structures. The phase differentiation process can be improved by collecting chemical information through X‐ray energy‐dispersive spectroscopy (XEDS) simultaneously with the crystallographic information through EBSD and then using the chemical information to pre‐filter the crystallographic phase candidates. This technique improves both the overall speed of the data collection and the accuracy of the final characterization. Examples of this process and the limitations involved will be presented and discussed.     

Morphology of the minipig kidney

The minipig has a multilobar kidney with a wide cortex and short papilla. The vascular bundles are of a simple type. Although short and long looped nephrons are both present, the short looped kind predominates. The minipig has many morphological similarities to dog and human kidneys. One particularly unique feature of the minipig papillary collecting duct cells, however, is the presence of electron-dense granules in the basal cytoplasm which appear to be secreted into the lateral intercellular spaces, perhaps forming a water-tight seal in a manner analogous to membrane-coating granules found in the epidermis of skin.     

Effects of probe pollutants on morphological and mechanical measurements of muscle and collagen fibers using atomic force microscopy

Because of the interaction between probes and samples, pollutants in buffer solution or in the air would easily bind to probes and make the probe polluted, which might influence the morphological and mechanical measurements with atomic force microscopy. The polluted probes might transfer the pollutants onto the samples and thus change the surface ultrastructure of samples, or collect the deviated feedback signals to make the phantasm images. The former process is irreversible even if a new probe is employed, and the latter one is a reversible process as long as changing the used/polluted probe. Effects of polluted probes on morphological and mechanical characteristics of insect flight muscle and rat tail tendon collagen I fibers had been discussed in this study, in which, we constructed a series of methods to avoid/reduce the collecting of phantasm images and deviated mechanical information, such as changing the scanning direction and scanning force, replacing the new probes, or cleaning the polluted probes. SCANNING 32:113–121, 2010. © 2010 Wiley Periodicals, Inc.     

Scanning electron microscopy of nerve fibers in human fetal cochlea

The cochleas of four human fetuses ranging 22–25 weeks gestation were studied by scanning electron microscopy (SEM) for the purpose of obtaining a better understanding of the nerve fiber arrangement in the human ear. After critical point drying, the specimens were dissected and the floor of the tunnel of Corti and the outer wall of Nuel's space were exposed for observation. Upper cochlear turns, especially the apical turn, seemed to be still immature. Observed nerve fibers were classified into three types:

• 1 Spiral fibers: Fibers traveling basalward and following the shape of the cochlea were found in both the tunnel of Corti and Nuel's space and believed to be the afferent nerves responsible for innervating the outer hair cells
• 2 Radial fibers: radiating outward from the osseous spiral lamina—one such radial fiber transversing high in the tunnel space (supposedly the efferent nerve servicing the outer hair cells), and another sort of radial fiber (found crossing the tunnel floor), the nature of which was uncertain.
• 3 Irregular fibers: Consisting of thin, randomly running fibers within the cochlea. The destination of these fibers was not determined, but possibly they represent transitory nerve branchings of afferent or more probably efferent nerves, which would later regress during maturation.

     

Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis

The pathogenesis of the pain in patellar tendon tendinosis ("jumper's knee") is unclear. We have recently presented new information about the sensory nervous system in the human patellar tendon, but there is very little information regarding the possible occurrence of a cholinergic system in this tendon. In the present study, specimens of pain-free normal tendons and chronically painful tendinosis tendons were examined by different immunohistochemical and histochemical methods. Antibodies against the M(2) receptor, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT) were applied, and staining for demonstration of activity of acetylcholinesterase (AChE) was also utilized. It was found that immunoreactions for the M(2) receptor could be detected intracellularly in both blood vessel cells and tenocytes, especially in tendinosis specimens. Furthermore, in the tendinosis specimens, some tenocytes were seen to exhibit immunoreaction for ChAT and VAChT. AChE reactions were seen in fine nerve fibers associated with small blood vessels in both the normal control tendons and the tendinosis tendons. The observations suggest that there is both a nerve related and a local cholinergic system in the human patellar tendon. As ChAT and VAChT immunoreactions were detected in tenocytes of tendinosis tendons, these cells might be a source of local acetylcholine (Ach) production. As both tenocytes and blood vessel cells were found to exhibit immunoreactions for the M(2) receptor, it is likely that both of these tissue cells may be influenced by ACh. Thus, in conclusion, there appears to be an upregulation of the cholinergic system, and an occurrence of autocrine/paracrine effects in this system, in the tendinosis patellar tendon.     

Progressive steps in the development of electron backscatter diffraction and orientation imaging microscopy

Ten years ago electron backscatter diffraction (EBSD) became available to a wider group active in materials research. This paper highlights some of the more significant developments in camera technology and software developments that have arisen since then. The use of slow‐scan charge couple device cameras for phase identification and rapid determination of orientation image micrographs is reviewed. The current limiting spatial resolution of the technique is shown to be less than 10 nm. A procedure for improving lattice spacing measurement by utilizing the full resolution of the camera is described with experimental measurements on silicon and nickel showing relative errors of plus/minus 3%. An investigation of partially recrystallized aluminium shows how the recrystallized fraction can be extracted with confidence but that the mapping of substructure in the highly deformed regions is questionable. Phase identification is described for complex cases in which the phase data tabulated in standard databases do not correspond to what is observed in the EBSD patterns. Phase mapping in a complex mineral in which chemical data and EBSD data are collected simultaneously is shown to be improved by recording both the chemical and the EBSD data into computer memory and proceeding with the phase discrimination and orientation measurement in off‐line analysis.     

Biochemical and morphological alterations in the Achilles tendon of mdx mice

Dystrophin‐deficient muscles have repeated cycles of necrosis and regeneration, being susceptible to injury induced by muscle contractions. Some studies have demonstrated that tendons are also affected in mdx mice, based especially on the changes in biomechanical properties arising from the respective linked muscles. However, most studies have focused only on alterations in the myotendinous junction. Thus, the purpose of this work was to study biochemical and morphological alterations in the Achilles tendons of 60‐day‐old mdx mice. Hydroxyproline quantification, showed higher collagen concentration in the mdx mice as compared with the control. No difference between the tendons of both groups was found in the noncollagenous proteins dosage, and in the amount of collagen type III detected in the western blotting analysis. The zymography for gelatinases detection showed higher amounts of metaloproteinase‐2 (active isoform) and of metalloproteinase‐9 (latent isoform) in the mdx mice. Measurements of birefringence, using polarization microscopy, showed higher molecular organization of the collagen fibers in the tendons of mdx mice in comparison to the control group, with presence of larger areas of crimp. Ponceau SS‐stained tendon sections showed stronger staining of the extracellular matrix in the mdx groups. Toluidine blue‐stained sections showed more intense basophilia in tendons of the control group. In morphometry, a higher number of inflammatory cells was detected in the epitendon of mdx group. In conclusion, the Achilles tendon of 60‐day‐old mdx mice presents higher collagen concentration and organization of the collagen fibers, enhanced metalloproteinase‐2 activity, as well as prominent presence of inflammatory cells and lesser proteoglycans. Microsc. Res. Tech. 78:85–93, 2015. © 2014 Wiley Periodicals, Inc.     

Ultrastructural demonstration of exocytosis in the intact rat adrenal medulla

Evidence is presented for morphological proof of exocytosis in the rat adrenal medulla in situ. Techniques were modified to allow perfusion of the intact adrenal gland with secretagogues (or electrical stimulation) followed by tannic acid. Unstimulated specimens demonstrated exocytotic (omega-shaped) profiles filled with flocculent material. This flocculation was also seen in the intercellular space. Stimulation of the adrenal medulla also resulted in the appearance of exocytotic profiles and an accumulation of the flocculent mass. This was often most evident in the subendothelial space. This is the first demonstration of exocytosis in the rat adrenal medulla by electron microscopy. The techniques used in this study will be useful for studying the pathway of secretory products of the adrenal chromaffin cell before they enter the vascular system.     

Protozoal infections in the acquired immunodeficiency syndrome

Several protozoa have emerged as the major opportunistic infections and cause of death in patients with acquired immunodeficiency syndrome (AIDS). Pneumocystis carinii pneumonia is the leading cause of death in AIDS patients. Electron microscopy (EM) usually shows numerous trophozoites and cysts of Pneumocystis filling up the entire alveolar space, while only cysts are seen under the light microscope. The focal thickening of cyst wall of Pneumocystis, as demonstrated by EM and manifested as a “parentheses” shaped structure with silver stain, serves as a diagnostic marker for Pneumocystis. Freeze-fracture EM has demonstrated the intimate contact between Pneumocystis trophozoites and the type I pneumocytes, which may contribute to the alveolar-capillary block, leading to severe respiratory distress. However, EM is seldom needed for the diagnosis of this infection. Toxoplasma encephalitis, which is an unusual clinical manifestation in cases of toxoplasmosis reported previously, has become a common complication and one of the major causes of death in patients with AIDS. Because subclinical infection by Toxoplasma is common, serologic tests usually offer no definite answers as to whether the infection is acute or chronic, active or past. The small size and its non-specificity in both morphology and tissue affinity make light microscopic diagnosis of toxoplasmosis difficult. Only immunologic staining, such as immunoperoxidase and immunofluorescence, can help to achieve a definite positive identification of the organism. When special antibodies or facility for such staining is not available, EM is the final resort for identifying Toxoplasma by showing the apical complex with the characteristic sausageshaped rhoptries. Cryptosporidiosis, practically unknown before the AIDS outbreak, has become one of the most common intestinal protozoa in both immunocompromised and immunocompetent patients. The protracted and sometimes fatal course of cryptosporidiosis in immunocompromised patients can be explained by the presence of autoinfective oocysts (thin-walled oocysts), as detected by EM, and by recycling of first-generation schizonts observed experimentally. While diagnosis of cryptosporidiosis can be made by detection of oocysts in stools in most cases, EM is still the last resort for a definitive identification of Cryptosporidium species. While the incidence of isosporiasis is still low, it has been found more frequently in patients with AIDS than in the general population. The parasite, Isospora belli, being a coccidian as is the Cryptosporidium species, is similar to the latter in its life cycle and clinical manifestations. However, the morphology of its diagnostic stage, the oocyst, is quite different from Cryptosporidium and it is much larger than the latter. The oocyst of Isospora belli, usually containing one sporoblast, can be detected by light microscopy in stools. Microsporidiosis, having been known only recently, is also relatively common in immunocompromised patients, including four patients with AIDS. Although this protozoan can be detected by light microscopy and its polar granules, identified by the periodic acid-Schiff or methenamine silver stain, are characteristic, a definitive diagnosis of microsporidiosis still depends on EM.     

Quantitative analysis of synapse structure with a semiautomatic interactive computer system: A study on identified pyramidal tract neurons in the sensory-motor cortex of cat

Pyramidal tract (Pt) neurons in the sensory-motor cortex of cat were labeled by injection of HRP into the spinal cord. Ultrastructural and quantitative analysis of the synaptic covering of their soma and apical dendrite (up to 100 μm from soma) was undertaken. We intercalated a visual-manual treatment between composite electron micrographs and a fully automated computer system and developed specific programs for evaluation of the morphometric data. Programs are included. A total of 412 synaptic boutons were examined that were found in contact with large Pt neurons. The mean linear percentage of the surface area covered by boutons was 26.2 ± 8.4% and the mean contacting length and cross-sectional area of the bouton profiles were 1.28 ± 0.58 μm and 0.89 ± 0.59 μm², respectively. All types of boutons with active zones accounted for 41.2% of the total. The distribution of two types of bouton (S- and F-type boutons, showing asymmetric and symmetric contacts, respectively) was examined quantitatively. The mean proportion of F-type boutons was 89.1% with a soma and S-type boutons contacting apical dendrites was 10.9%. In addition, GABAergic boutons were identified with the soma by immunocytochemistry with antibodies against glutamic acid decarboxylase. They formed symmetric synaptic contacts with the Pt cells that were identical to those formed by F-type boutons. The quantitative analysis revealed that synaptic clefts are narrower and synaptic vesicles are smaller in symmetric F-type boutons than in S-type boutons forming asymmetric contacts. These data establish that at least three parameters (postsynaptic density, synaptic cleft, and size of vesicles) can be utilized singly or in combination to identify GABAergic inhibitory synapses in neocortex.     

A computer program for the systematic sampling of sections in microscopic morphometry

A computer program is described that facilitates systematic and unbiased sampling in morphometry. Input data are coordinates of the four corners of a section as displayed in the position indicators of the microscope stage. Outputs are coordinate values in the form of a regular lattice that describes where to place the stage and perform the sampling on the section. In addition, some other data are provided by the program, such as total area of section, length of sides, etc. The program is written in BASIC but can be easily converted to other computer languages.     

Scanning force microscopy of chromatin fibers in air and in liquid

We have adapted specimen preparation techniques of conventional electron microscopy for visualizing chromatin structures in the scanning force microscope (SFM) in air and in liquid. The beaded substructure of the nucleoprotein filament was obtained after hypotonic lysis of chicken erythrocytes and air drying, whereas supranucleosomal structures were preserved after treatment of cell nuclei with detergent. In the latter case, the nucleosomes were still distinct but appeared more condensed. A modified droplet diffusion-spreading technique of chromatin from Namalwa cells (a human B-lymphoid line) yielded a uniform filamentous morphology and similar fiber appearance. A reversible swelling of spread chromatin was observed upon exposure of air-dried samples to solutions differing in salt concentrations.     

A microcomputer program in “BASIC” for the accurate determination of the height of small particles

A formula is derived to enable the calculation of the true height of an object, such as a shadowed latex bead, from electron micrographs. Knowing only the angle of shadowing and the length of the evaporated shadow, and by substituting these values in the derived formula, a microcomputer may be programmed to carry out the necessary computations. An example of such a microcomputer program is given. The correct determination of the height of particles by electron microscopy using the shadowing technique is one of the most accurate methods available for the determination of small particle height.     

Cholinergic synapses in the central nervous system: Studies of the immunocytochemical localization of choline acetyltransferase

Cholinergic synapses can be identified in immunocytochemical preparations by the use of monoclonal antibodies and specific antisera to choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker for cholinergic neurons. Electron microscopic studies demonstrate that the fibers and varicosities observed in light microscopic preparations of many brain regions are small-diameter unmyelinated axons and vesicle-containing boutons. The labeled boutons generally contain clear vesicles and one or more mitochondrial profiles. Many of these boutons form synaptic contacts, and the synapses are frequently of the symmetric type, displaying thin postsynaptic densities and relatively short contact zones. However, ChAT-labeled synapses with asymmetric junctions are also observed, and their frequency varies among different brain regions. Unlabeled dendritic shafts are the most common postsynaptic elements in virtually all regions examined although other neuronal elements, including dendritic spines and neuronal somata, also receive some cholinergic innervation. ChAT-labeled boutons form synaptic contacts with several different types of unlabeled neurons within the same brain region. Such findings are consistent with a generally diffuse pattern of cholinergic innervation in many parts of the central nervous system. Despite many similarities in the characteristics of ChAT-labeled synapses, there appears to be some heterogeneity in the cholinergic innervation within as well as among brain regions. Differences are observed in the sizes of ChAT-immunoreactive boutons, the types of synaptic contacts, and the predominant postsynaptic elements. Thus, the cholinergic system presents interesting challenges for future studies of the morphological organization and related function of cholinergic synapses.     

Viral infections in the acquired immunodeficiency syndrome

The following communication is a tripartite synopsis of the role of viral infection in the acquired immunodeficiency syndrome (AIDS). The first section describes the impact of viral opportunistic infection in AIDS; for each virus, clinical presentation and diagnosis, laboratory diagnostic approaches (with emphasis on electron microscopy), and therapeutic interventions attempted to date are discussed. The second segment explores current theories on the pathogenesis of AIDS, and describes diagnostic and therapeutic approaches to the syndrome itself. The final section catalogues ultrastructural anomalies in the cells of AIDS patients, many of which have been mistakenly identified as etiologic agents.     

Molecular genetics and structure of the human immunodeficiency virus

A novel human lymphotropic virus capable of crippling the immune system by infecting and destroying T4 antigen-positive cells is now known to be the etiologic agent of the acquired immune deficiency syndrome (AIDS). The AIDS or human immunodeficiency virus (HIV) belongs to a family of RNA viruses called retroviruses. Several strains of HIV have been molecularly cloned, and DNA sequence comparisons have established that the proviral DNA genome is 9.7 kilobase pairs. The genome possesses characteristic retrovirus features including structural genes, flanked by long terminal repeats, in the order gag, pol, and env and, in addition, four unique nonstructural genes, several of which appear to be essential in regulating virus replication. Electron microscopy has played an important role in elucidating structural, genetic, and molecular properties of HIV and has aided in its classification as a member of the Lentivirnae retrovirus subfamily. Heteroduplex mapping methodologies pertinent to these findings are described. Although the relationships show considerable divergence, the similarities between HIV and lentiviruses are profound and encompass an indistinguishable morphology, genome sequence homology and topography, genomic diversity, and overlapping biology, including a preference for infecting cells of the immune system, a cytopathic effect in vitro, and the ability to produce a persistent, slowly progressing, degenerative disease in vivo. The newest HIV class (HIV-2) has recently been molecularly characterized. HIV-2 also bears all the hallmarks of a lentivirus but is more closely related to simian immunodeficiency viruses than the previously described HIV-1, despite a similar biology. The HIV-lentivirus phylogenetic relationship has broad implications for the AIDS disease process and has given new importance to the study of the natural history and pathogenesis of animal lentiviruses in searching for clues to prevent the spread of AIDS.