Structure of mitochondria and vacuoles of Candida utilis and Schizosaccharomyces pombe studied by electron microscopy of serial thin sections and model building

The structure of mitochondria and of vacuoles in Candida utilis and Schizosaccharomyces pombe has been studied by electron microscopy of serial thin sections and subsequent model building. The models of the two cells of C. utilis which were studied confirmed our earlier findings, made by high voltage electron microscopy of thick sections, that there is a single, branched and continuous mitochondrial network in the cell (Davison & Garland, 1975). A model of a S. pombe cell showed that the mitochondrial structure was far more continuous than expected from inspection of thin sections, there being but two large and two small mitochondria. The models demonstrated that the few large vacuoles in C. utilis were interconnected into a single cluster, whereas in S. pombe there were two separate complexes of interconnected vacuoles towards each pole of the cell.     

The chondriome of selected trypanosomatids. A three-dimensional study based on serial thick sections and high voltage electron microscopy

The unitary nature of the chondriome of two species of trypanosomatids, Blastocrithidia culicis and Trypanosoma cruzi, has been demonstrated by utilizing serial thick-sectioning techniques combined with high voltage electron microscopy. Profiles of mitochondrial elements seen in thin sections and suspected to be parts of a continuum were confirmed by serial thick sectioning (0.25-0.50 mum thick) and stereopair analysis to be parts of the same mitochondrion. Three-dimensional models obtained from tracings of mitochondrial profiles on cellulose acetate reveal the mitochondrion of B. culicis to consist of a posterior mass with six tubular extensions extending upward and terminating in the anterior apex. The kinetoplast was found suspended between two of the tubular extensions, or less frequently, protuding as a nodule from one of the extensions. A bifurcation of one of the extensions was found in some specimens. The mitochondrion of T. cruzi consists of a triangular- shaped convoluted tubule, the base being the kinetoplast portion while the apex is directed posteriorly. The mitochondrion bifurcates behind the flagellar pocket, lateral to the kinetoplast, sending two entwined extensions into the tenuous anterior apex. Whether the mitochondrion of T. cruzi is unitary in the trypomastigote form was not determined in this study, since only epimastigote forms were used.     

Electron microscopy of thin sections of Candida utilis: The structure of the cell wall

Summary Details of the structure of the Candida utilis cell wall were described. Using intact cells, cell walls, about 0.02 m thick, have been resolved into three electron dense layers, each about 700 Å thick, made up of materials of very similar electron opacity. The central and the inner layers of the wall seem to be closely packed forming a slightly more compact structure of somewhat greater electron density. Laminations were observed in some of the inner layers of the sections, particularly in some areas where the cell wall had separated: The possibility of this lamination being associated with the presence of chitin in the framework of the cell wall is discussed. Interesting observations have also been made in the sections of the heat-killed cells confirming the existence of a central electron dense cell wall layer, differing from the inner and the outer ones. Underlying the cell wall is the cytoplasmic membrane, a not too well defined sinous structure with some invaginations.     

Internal structure of ovomacroglobulin studied by electron microscopy.

As a model for the molecular structure of proteins belonging to the alpha 2-macroglobulin family, ovomacroglobulin of reptilian origin was studied by electron microscopy in the original tetrameric form as well as in the dissociated forms into half- and quarter molecules. The following aspects of the molecular internal structure which had previously not been known for the homologous human alpha 2-macroglobulin or chicken ovomacroglobulin were revealed. First, the negatively stained tetrameric native protein gave an appearance of a collection of four semi-circular strings placed on the four corners of a molecule. They were connected to each other in the center of a molecule through a set of globular domains which formed a cross-figured subunit contact region. Second, two kinds of active half-molecules prepared either by the reduction of intersubunit disulfide bonds or by the disruption of noncovalent subunit interface had similarly elongated forms having semi-circular units on the two ends, indicating quasi-equivalent subunit arrangement in the two kinds of half-molecules. We thus concluded that the structure of native ovomacroglobulin can be represented by four circular strings each equipped with an extra domain to form the central intersubunit contact region. The results may also be adapted to the internal structure of human alpha 2-macroglobulin because it was sometimes possible to observe similar ring-like internal structure in the human protein.     

A method for staining actin-containing structures in thick plastic sections for medium voltage electron microscopy

A staining procedure has been developed for imaging actin-containing structures in thick plastic sections in the electron microscope. The stress fibres of a fibroblastic cell line were used as a model system, and were first characterized immunocytochemically. After fixation of cells in formaldehyde, mordanting in a solution of gadolinium chloride allows stress fibres to be stained for light microscopy with haematoxylin. A brief exposure to a solution of ammonium paramolybdate renders haematoxylin-stained structures sufficiently electron-dense to be imaged in 1 micron thick plastic sections in a JEOL 200CX electron microscope, operating at 200 kV, and possibly in conventional instruments operating at 100 kV, particularly if equipped with a lanthanum hexaboride source.     

Mitochondrial structure revealed by high-resolution scanning electron microscopy

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.     

Tri-dimensional morphometric analysis of astrocytic processes with high voltage electron microscopy of thick Golgi preparations

A characteristic feature of the astrocytic processes is to assume the form of shin sheets or lamellate coverings of other brain constituents. We analyzed the extensive and finely divided processes of the protoplasmic astrocyte in the molecular layer of the rat dentate gyrus by means of computer electron tomography and stereo-photogrammetry using tilted high voltage electron microscope images of thick Golgi preparations. The surface area and volume of the astrocytic processes were measured and the surface/volume ratios were estimated. The surface/volume ratios of astrocytic processes in the neuropile ranged from 18.9 to 33.0 per μm, and the mean value was 26.2 ± 5.0 per μm. The values were roughly comparable to those previously reported for the microdomain of Bergmann glia cell terminal processes in the rat cerebellum, which were estimated from reconstructions using thin serial section electron microscope images. The large surface to volume ratio of the astrocytic processes in the neuropile resulted from the lamellar nature of the processes interposed between other cellular elements, and may reflect the functional activities of the astrocyte. The results suggest the usefulness of the electron tomography and stereo-photogrammetry for three-dimensional morphometrical analysis of the astrocytic processes, although both techniques can be expected to be refined further in order to provide more precise measurements of these complicated processes.     

The quatenary structure of Pseudomonas cytochrome oxidase studied by electron microscopy.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) was studied by negative staining in the electron microscope. The best resolution was obtained with uranyl oxalate (pH 6.0) as negative stain. Electron micrographs confirm the idea of the dimeric structure of the enzyme. A rough model of cytochrome oxidase was constructed based on different projections of the molecule seen in the electron micrographs. In this model the subunits are identical and sterically equivalent.     

The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.     

Sewage coliphages studied by electron microscopy.

Sewage was enriched with 35 Escherichia coli strains, and sediments of enrichment cultures were studied in the electron microscope. They contained up to 10 varieties of morphologically different particles. T-even-type phages predominated in 14 samples. Thirteen phages were enriched, representing the families Myoviridae (seven), Styloviridae (two), Podoviridae (three), and Microviridae (one). Twelve of these corresponded to known enterobacterial phage species, namely, 121, K19, FC3-9, O1, 9266, T2, 16-19, kappa, beta 4, N4, T7, and phi X174. Cubic RNA phages and filamentous phages were not detected. Types 121 and 9266 have previously been observed only in Romania and South Africa. Identification by morphology is usually simple. Our investigative technique is qualitative and will not detect all phages present. Most enrichment strains are polyvalent, and electron microscopy is always required for phage identification. In a general way, electron microscopy seems to be the method of choice for investigation of phage geography and ecology.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

Dimeric arrangement and structure of the membrane-bound acetylcholine receptor studied by electron microscopy.

The acetylcholine receptor protein (AChR) from the electric organ of Torpedo marmorata is studied in its membrane-bound form by electron microscopy and single-particle image averaging. About half the molecule protrudes from the membrane surface by approximately 5 nm. The low-resolution 3-D structure of this hydrated portion, including its handedness, can be deduced from averaged axial and lateral projections and from freeze-etched membrane surfaces. In native membrane fragments, a dimeric form of the AChR is observed and the relative orientation of the AChR monomers within the dimer is established. The dimers disappear upon disulfide reduction of the membrane preparations, whereas the average axial projections of the AChR monomer remain unaffected. Since the existence of disulfide bonds linking AChR monomers between their respective delta-subunits is well documented, the approximate position of the delta-subunit within the low-resolution structure of the AChR molecule can be deduced from the structure of the dimers.     

A method for obtaining histological sections from tissue previously studied by scanning electron microscopy

An accelerated method of paraffin embedding of tissue specimens previously examined with scanning electron microscopy is proposed aimed to obtain sections for routine histological examination. The tissue is passed through acetone, absolute alcohol, alcoholic-oil celloidin solution, chloroform to be eventually mounted into paraffin. The method allows obtaining good quality sections within 24 hours.     

Applications of high voltage electron microscopy to botanical ultrastructure

Although high voltage electron microscopes have been in general use over the past decade microscopists have tended to ignore the contribution their use could make to the study of plant ultrastructure. The majority of biological high voltage research has been restricted to the fields of zoology and bio-medicine.The high voltage electron microscope (HVEM) has several advantages over the conventional transmission electron microscope (CTEM) when applied to biological specimens. These include increased penetrating power of the electron beam, reduced chromatic abberation in thick specimens, and both reduced beam heating and ionization damage. All these factors permit the observation of thick sections, whole cells and hydrated specimens. Most botanical HVEM research has been restricted to the study of thick sectioned material. Various staining techniques have been applied to overcome the decrease in image contrast at high accelerating voltages, but the commonest have been modifications of lead and uranium stains previously developed for thin sections. Selective staining can simplify the mass of information in a thick specimen thus specific structures may be studied against an unstained background. Acidified phosphotungstic acid can be used to stain the plasma membrane and osmium impregnation will selectively stain many of the cytoplasmic membranes in a variety of specimens. Other techniques for the selective localization of cell components, such as enzyme cytochemistry and autoradiography have yet to be fully exploited by high voltage electron microscopists.Interpretation of the great quantity of information in a thick specimen can be facilitated by tilting the specimen and producing stereo pairs. Quantitative depth information can be extracted from stereo pairs by the use of measuring mirror stereoscopes or by direct measurement from each member of a stereo pair. Serial thick sectioning has been employed as an alternative to prolonged serial thin sectioning to aid in the reconstruction of large specimens.Stereo images can be viewed in a variety of ways with lenticular pocket stereoscopes, reflecting mirror stereoscopes, prismatic spectacles, polarized spectacles when projected onto a non depolarizing screen or presented on TV monitors.     

Submicroscopic organization of M. pneumoniae studied by means of negative contrasting, ultrathin sections and scanning electron microscopy]

The ultrastructure of M. pneumoniae, grown on a solid culture medium and in a liquid one, was studied by a number of methods. Two types of cells were shown to prevail in the culture: spherical cells (0.5--1 micrometer) forming chains of different configurations and filamentous cells (5 micrometer long and greater) with spherical enlargements along their whole length. The absence of microcapsules made M. pneumoniae different from other species of mycoplasms, and the organism proliferated by division into 2 daughter cells, equal or unequal in size, by the segmentation of the cytoplasm and the formation of elementary bodies inside the cell and on its surface.