Conventional Thin-section Electron Microscopy Research Articles

Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures

SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.

Assembly of Human Immunodeficiency Virus Precursor Gag Proteins

To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 A is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers.

Cytoskeletal organization in the neurohypophysial axons of rats

Cytoskeletal organization in axons of the rat neurohypophysis was examined by quick-freeze deep-etch electron micrscopy and conventional thin-section electron microscopy. In the neurohypophysial axons, neurotubules and neurofilaments were arranged almost in parallel with each other, and interlinked by cross-bridges. Because the localization of MAP1A on the axonal cytoskeleton was shown by immunoelectron microscopy, MAP1A is a component of the cross-bridges between neurotubules. The neurotubules and neurofilaments occupied their respective domains in the axon. The neurotuble:neurofilament ratio was variable axon by axon. In some axons, neurotubules overwhelmed neurofilaments in number. In other axons, neurofilaments were predominant, and neurotubules were few. The neurosecretory granules were located in association with either the neurotubule or neurofilament domain. Fine fibrils 5–10 nm in diameter were associated with the inner surface of the axonal plasmalemma. In the Herring body, a few neurotubules occurred in the marginal region while neurofilaments were rarely seen, indicating that most of the neurosecretory granules have no relationship with the neurotubules in the body. These results suggest that the cytoskeleton in neurohypophysial axons may be involved in regulation of axonal transport and storage of neurosecretory granules.

Grid-mapped freeze-fracture analysis of gap junctions in gray and white matter of adult rat central nervous system, with evidence for a "panglial syncytium" that is not coupled to neurons.

In white matter regions of the brain and spinal cord of adult mammals, gap junctions previously were observed linking astrocytes to astrocytes, as well as to oligodendrocytes and ependymacytes. The resulting "functional syncytium" was proposed to modulate the ion fluxes that occur during electrical activity of the associated axons. Gap junctions also have been reported linking neurons with glia, and functional neuronal-glial coupling has been postulated. To investigate the glial syncytium and the neuron-to-glial coupling hypotheses, we used "grid-mapped freeze fracture," conventional thin-section electron microscopy, and light microscope immunocytochemistry to examine and characterize neurons and glia in gray and white matter of adult rat brain and spinal cord. We have obtained quantitative evidence for the abundance and widespread distribution of gap junctions interlinking the three primary types of macroglia throughout both gray and white matter of the mammalian central nervous system (CNS), thereby extending the concept to that of a functional panglial syncytium. In contrast to previous reports, we show that of more than 400 gap junctions in which both participating cells were identified, none were between neurons and glia. Thus, neuronal coupling and glial coupling involved separate and distinct pathways. Finally, putative water channels (i.e., "square arrays") were confirmed to be abundant and in close association with gap junctions in astrocytes and ependymacytes. Because the astrocyte "intermediaries" extend cytoplasmic conduits throughout gray and white matter of brain and spinal cord, from the ependymal layer to the pia-glial limitans, and from oligodendrocytes surrounding axons to astrocyte endfeet surrounding capillaries, the proposed panglial syncytium, with its abundance of water channels and intercellular ion channels, is optimally positioned and equipped to modulate water and ion fluxes across broad regions of the CNS.

3-D analysis of cellular lipid metabolism: Reconstruction of the tubular network

A predominant feature of early atherosclerotic lesions is the presence of macrophages and smooth muscle cells in which large quantities of lipid have accumulated. This lipid occurs within both small, cytoplasmic, cholesterol ester droplets and large, lipid-swollen secondary lysosomes. Time resolved studies suggest lysosomal engorgement promotes the disease progression. Using cultured cells we have shown that lysosomal engorgement in smooth muscle cells is accompanied by the elaboration of an extensive network of tubules. The tubules are rich in acid hydrolases and appear to be an extension of the trans Golgi network. As part of our effort to understand the causes and significance of lysosomal lipid accumulation we have undertaken to characterize the extent and development of the tubular network. The tortuous nature of the network makes it difficult to visualize by conventional thin section electron microscopy. Thus, intermediate voltage electron microscopy of thick (1-3 um) sections in which the tubules have been contrasted using acid phosphatase cytochemistry has been essential to these studies. Cultured rabbit smooth muscle cells were incubated with sonicated lipid droplets composed of 72% cholesteryl oleate, 25% triolein, and 3% phospholipid. Prior to incubation, cells had no visible lipid stores and 92% of cells showed no acid phosphatase-containing tubules. In cells with tubules, the tubules occupied less than 0.1% of the cell volume. In contrast, after 4 h of exposure to droplets 81% of cells showed lysosomal lipid accumulation with 67% exhibiting extensive, interconnecting, acid phosphatase positive tubular networks. Although stereo-pair images provided much information about the extent of the network (figure 1), the exact relationship of the network to lipid-engorged lysosomes was somewhat ambiguous due to superposition of objects. In order to clarify the relationship of network to lysosome, a tomographic reconstruction was computed from an IVEM tilt series encompassing +/− 60° in 2° increments. Images were digitized using a DAGE model 81 video camera. Rotational alignment using fiduciary markers was accomplished using the algorithm described by Lawrence. Translational alignment and weighted back-projection reconstruction were carried out as described by McEwen. Volumes were rendered using a PIXAR computer graphics workstation. The reconstruction (figure 2) revealed that extensions of the network made contact with the lipid-swollen lysosomes suggesting that the network might transfer hydrolases directly to secondary lysosomes.

The association of microtubules with an acid phosphatase-containing tubular compartment in macrophages.

Received for publication October 8, 1991 and in revised form January 10, 1992 Resident peritoneal murine macrophages typically have acid phosphatase (AcPase) activity in lysosomes which are spherical in shape and have a perinuclear distribution. Upon stimulation with certain phorbol esters there is a rapid and dramatic modification of the AcPase-containing compartment into tubular structures which are often interconnected. The formation and maintenance of these tubular structures are dependent upon the presence of an intact microtubule system. Treatment of cells with the microtubule-depolymerizing agent nocodazole blocks the formation of the tubular structures and causes these tubular compartments to collapse toward the nucleus when nocodazole is administered after they are formed. We have employed whole mount electron microscopy and enzyme cytochemistry to document the association of the phorbol ester-induced AcPase-containing compartment and cytoplasmic microtubules. This methodological approach has the advantage of increased resolution when compared to optical microscopy and of an improved vantage point for analysis when compared to conventional thin section electron microscopy.

Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections.

We describe two methods for staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thin-section electron microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy. In the first method of immunostaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying. In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity.

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.

Three‐dimensional structure of astrocytes in the rat dentate gyrus

Structural features of astrocytes in the rat dentate gyrus were studied by means of light and high-voltage electron microscopy of Golgi-impregnated materials, conventional electron microscopy, combined Golgi-electron microscopy, and immunohistochemistry for glial fibrillar acidic protein. Astrocytes in the dentate gyrus were of the protoplasmic type and were classified into six subtypes based on the location of their somata; i.e., astrocytes in the polymorph layer, in the subgranular zone, in the granular cell layer, at the border of the granular cell layer and the molecular layer, in the molecular layer, and subjacent to the pia surface. Stereoscopic observations of 5-micron-thick sections of Golgi-impregnated materials revealed three-dimensional structural details of astroglial processes not apparent in either the light microscope or in conventional thin-section electron microscopy. Most of them were basically thin sheets, varying in shape and size in accordance with their sites. In the granule cell layer, thin veillike sheets or lamellae, originating from three kinds of astrocytes (subtypes 2, 3, and 4), intervened between granule cell somata, whereas in the plexiform and molecular layers small leafletlike appendages originating from astrocytes (subtypes 1, 2, 4, and 5) intermingled with one another, making spongelike conglomerates. Thus astrocytes in the subgranular zone and those at the border of the granule cell layer and the molecular layer showed prominent regional differentiation of their processes among layers. In addition to these sheetlike processes, thin threadlike processes were also common.

Cytoplasmic virus-like particles associated with the nucleolus in wheat, similar to ?S bodies? and retroposon particles

Wheat roots from germinating seedlings of Chinese spring wheatTriticum aestivum grown for 36 hours at 20°C were examined by conventional thin-section electron microscopy. Virus-like particles were seen inside a large cytoplasmic intrusion into the nucleus having the appearence of a nucleolar vacuole. The particles were isometric and about 50 nm in diameter with a membrane-like coat and a small core. The cytoplasmic intrusion was bounded by nuclear envelope with pores apparent where it abutted nucleoplasm. The particles are similar to previously reported solitary particles “S bodies” from a range of plants but are also similar in size and morphology to the retroposon particles associated with copia like elements in other organisms. The position of the virus-like particles in the young wheat roots is discussed in relation to interactions with components of the cell skeleton.

Fine structural aspects of phagocytotic activity in inverted follicle cells of cultured porcine thyroids.

Inversion of thyroid follicles took place when they were isolated by collagenase and trypsin and cultured in suspension in Eagle's medium supplemented with 10% fetal calf serum without TSH. The apical surface facing the culture medium contained numerous microvilli and a central cilium, while the luminal surface became flattened. Phagocytotic activity by pseudopods was promoted after addition of TSH to the culture medium. When the inverted follicles were incubated in culture medium containing TSH (50 mU/ml) and human red blood cells, or TSH and polystyrene latex beads (2.02 micron in diameter) for 1-3 h, numerous red blood cells or latex beads respectively were observed to be taken up by the epithelial follicle cells by scanning electron microscopy, as well as conventional thin-section electron microscopy. These results show that the apical surface (culture medium side) of the epithelial cell of the cultured thyroid follicle whose polarity is reversed phagocytoses red blood cells and latex beads.

7] Membranes of pancreatic zymogen granules

Zymogen granules (ZG) of pancreatic acinar cells are large spherical cytoplasmic organelles, where digestive enzymes are stored in concentrated form prior to their release to the extracellular space. Owing to the large size of granules, its cumulative surface area is much smaller than that of the other cytoplasmic membrane-bound compartments. In conventional thin-section electron microscopy, the ZG membrane reveals no distinctive features. In freeze-fracture, it is characterized by an extremely low density of intramembrane particles, most of which are attached to the protoplasmic fracture face. Zymogen granule membranes are not isolated directly from tissue homogenates but are obtained by subfractionation of purified granules. The purification of ZG took advantage of the fact that these organelles, when centrifuged into a pellet, tend to stick tightly to each other at the bottom of tubes, and thus to separate from other sedimented organelles, which are recovered in more superficial layers of the pellet. Under carefully controlled centrifugation conditions and provided that the ZG concentration in the starting preparation is large, a highly purified fraction can be obtained.

Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

Cell surface specializations and intercellular junctions in human amniotic epithelium: an electron microscopic and freeze-fracture study.

Cell surface specializations and intercellular junctions of human term amniotic epithelium were examined by conventional thin-section electron microscopy, after staining with the cationic probes ruthenium red and cationic ferritin, and by freeze-fracture methods. Desmosomes were the predominant type of intercellular junction and often the most apical of the junctional types. In freeze-fracture replicas, desmosomes were characterized by roughly circular areas of large, often irregular, P-face intramembranous particles. Gap junctions were identified in the laterobasal regions between cells. In thin sections they were characterized by a narrow intercellular space, and in freeze-fracture replicas had a typical plaquelike arrangement of P-face intramembranous particles and E-face depressions. Hemidesmosomes at the basal cell surface were characterized by occasional large particles and clusters of particles on both the E and P fracture faces. No evidence of tight junctions was found. The apical cell surface was heavily stained by both ruthenium red and cationic ferritin, indicating the negatively charged nature of this surface. Ruthenium red penetrated between the epithelial cells and bound to anionic materials on the lateral cell surfaces, especially at the location of desmosomes. Below the base of the intercellular cleft, large ruthenium red-positive granules were present in the extracellular matrix. The possibility that the anionic substances in the intercellular region may contribute to the control of permeability in the amniotic epithelium is discussed.

Fenestrae in the rough endoplasmic reticulum of Xenopus laevis hepatocytes.

The rough endoplasmic reticulum (RER) of Xenopus laevis hepatocytes was examined by freeze-fracture and by conventional thin section electron microscopy. Much of the RER was present as stacks of cisternae at the cell periphery but, in addition, large whorls of cisternae were seen in the cytoplasm in most sections. Freeze-fracture replicas revealed fenestrae in both stacked and whorled cisternae, although the fenestrae were more numerous in the whorls. The role of these fenestrae is unknown, but such structures would facilitate access of precursors to the protein synthetic machinery in this highly metabolically active cell type. This would be particularly important in RER whorls, where the innermost cisternae would otherwise be isolated from the rest of the cytoplasm.

Stereoscopic Visualization of Selectively Stained Neuromuscular Junctions Using High Voltage Electron Microscopy

High voltage electron microscopy (HVEM) combined with selective histochemical staining yields information otherwise difficult to obtain. Such selective staining follows the irregular contours, channels or surfaces of the structures being studied and thus is not easily traced in conventional thin section electron microscopy. Often the amount of enzyme activity is insufficient to provide an accurate estimate of quantitative differences in reaction product between experimental and control tissues. By taking stereo pairs of micrographs of serial thick sections at 1 MeV it is possible to obtain precise localization of very electron dense reaction products at high resolution and to view the three-dimensional aspects of enzyme distribution. Using stereoscopic visualization a cholinesterase reaction product is identified in neuromuscular junction regions isolated from the hind limb muscles of a dystrophic strain of mice.

Studies of excitable membranes. I. Macromolecular specializations of the neuromuscular junction and the nonjunctional sarcolemma.

The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100-110-A particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110-140-A particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-A particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol.63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.