Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.     

Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy

An atypical peg-like terminal constriction (“peg”) on metaphase chromosomes of the plant genus Oziroë could be identified as a nucleolus organizing region (NOR) by detecting 45S rDNA with correlative light microscopy (LM) and scanning electron microscopy (SEM) in situ hybridization (ISH). Using high-resolution 3D analytical SEM, the architecture and DNA distribution of the peg-like NOR were characterized as typical for chromosomes, albeit with significantly smaller chromomeres. ISH procedure was improved for SEM concerning signal localization, labeling efficiency, and structural preservation, allowing 3D SEM analysis of the peg-like NOR structure and rDNA distribution for the first time. It could be shown that implementation of FluoroNanogold markers is an attractive tool that allows efficient immunodection in both LM and SEM. A model is proposed for the peg structure and its mode of condensation.     

Structure of trichomonads as revealed by scanning electron microscopy.

A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné, Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal; emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

Exocytosis in human salivary glands visualized by high-resolution scanning electron microscopy

The luminal membrane of salivary acinar cells creates a specialized cell surface area that accepts exocytosis and undergoes dynamic changes during secretion. These changes were visualized three-dimensionally from both the inside and outside of the cell in human parotid and submandibular glands, by application of in vitro secretory stimulation and then of OsO₄ maceration to remove cytoplasmic organelles by varying degrees. In control glands treated without secretagogues, the luminal surface of serous acinar cells bore well-developed microvilli with only an occasional incidence of exocytotic profiles. Following treatment with the β-adrenergic agonist, isoproterenol, considerable shortening and loss of microvilli occurred along the luminal membrane where, on its cytoplasmic side, many protuberances of sizes similar to or smaller than those of single secretory granules (～1 μm in diameter) appeared. The cytoplasmic surface of these protuberances exhibited small vesicles (～100–150 nm in diameter) that, by transmission electron microscopy, were shown to be coated pits or vesicles present on or around the exocytosed granule membranes. Treatment of tissues with the muscarinic agonist carbachol also caused a decrease of microvilli and the appearance of protrusions at the luminal membrane. However, unlike isoproterenol treatment, many of these protrusions were devoid of small pits or vesicles and were much larger than a single secretory granule. These results indicate that (1) secretory stimulation causes the dynamic transformation of microvilli at the luminal membrane, where granule docking and membrane fusion take place, and (2) after fusion, the exocytosed membranes are processed differently, by coated pit/vesicle mediated or non-mediated mechanisms, according to the autonomic receptor control.     

The maxillae of Drosophila melanogaster as revealed by scanning electron microscopy

The scanning electron micrographs show the external morphology of the maxillae of Drosophila melanogaster. Specifically, they illustrate the patterning of the different types of chemo-receptive sensilla on the maxillary palpi making possible a clearer understanding of the structure of the tiny maxillary lobes. It appears that the maxillary lobes act as “cleaning brushes” during the feeding process.     

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

Summary The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.     

New characters in the Gnathostomulid mouth parts revealed by scanning electron microscopy

An analysis with SEM of the mouth parts of 16 species belonging to 10 genera of Gnathostomulida resulted in the following new characters and conclusions: 1) At least in the genus Haplognathia, jaw teeth that are visible by conventional light microscopy are composed of the same aggregated needle-like denticles that are found, often in large numbers, on the basal plates of many filospermoid species. 2) Other new ultrastructural tooth features include the tricuspid basal plate teeth in Problognathia minima, tripartite teeth in Austrognathia and Austrognatharia, and the clear separation, in the Gnathostomula basal plate, of a mediodorsal set of teeth from a more extensive rostroventral set. 3) Three rows of teeth, as typical for Gnathostomulidae and Austrognathiidae, are also present in the filospermoid Haplognathia filum. 4) The wide range of geographic variation in Haplognathia ruberrima is confirmed by significant differences in jaw teeth between specimens from Belize and Bermuda. 5) A compartmentalized involucrum is present in Labidgonathia longicollis. 6) A pair of lamellae addentales, until now only known from Valvognathia pogonostoma, was found in Tenuignathia rikerae, Problognathia minima, and probably also Labidognathia rikerae. 7) In all gnathostomulids, the lamella symphysis is composed of identical rods that are considered homologous with those in the mouth parts of Rotifera and Micrognathozoa.     

The fine structure of nuclei as revealed by electron microscopy

Summary The process of nucleolus formation has been studied by electron microscopy in spermatogonia of new-born, 15-day-old mice. One of two heteropycnotic sex chromosomes is concerned with nucleolus formation in the type A spermatogonia. The evidence for such formation has been presented with regard to behaviour and fine structure of both sex chromosome and nucleolus, Nucleolar material appears at one of two heteropycnotic sex chromosomes which are closely attached to the nuclear envelope. The two sex chromosomes approach each other, and subsequently one of them migrates into the central part of the nucleoplasm, being related to the nucleolar material which develops to show a nucleolar configuration. The sex chromosomes are homogeneously electron dense during the nucleolus formation, but assume a vesicular form at the middle stage of its development. The nucleolus is mostly of fibrillar and amorphous components at early stages of its development, but the granular components increases in amount as development proceeds. The final, mature nucleolus is composed of irregularly twisted nucleolonemata consisting of granular components, separated from fibrillar and amorphous areas. The compactly dense sex chromosome remains closely connected with the mature nucleolus.     

A critical reevaluation of the structure of the rat uriniferous tubule as revealed by scanning electron microscopy

Summary The fine structure of luminal surface of clearly identified portions of uriniferous tubules has been studied by scanning electron microscopy to elucidate some controversies concerning the topography of certain surface formations. The results show a characteristic pattern of the luminal surface in the region of Henle's loop, which was assumed by previous authors, to belong to the collecting tubule. Furthermore it is demonstrated that no cilia are present within the terminal portion of the collecting tubules.     

Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

Origins of prehistoric flints: The neocortex memory revealed by scanning electron microscopy

Flint has been widely used during prehistoric times and the question of determining the origin of specific pieces is recurrent in archaeological discussions. We have run a scanning electron microscope examination of the surfaces of both natural flints from geological deposits and flaked artefacts excavated from prehistoric sites in the Massif Central (France). These samples exhibit mineralogical textures resulting from genomorphic and phenomorphic processes, dispersed alteration in the form of erosion as well as the dissolution and migration of more or less mobile elements. Rocks used to manufacture artefacts at Sainte-Anne 1 (Haute-Loire, France) and Payre (Ardèche, France) show various kinds of alteration related to the range of phases of transport and deposition to which they were subjected, beginning with their first exposure in a geological context and following through to the post-depositional phase, (i.e., following abandonment as artefacts in an archaeological site). Later phenomena do not however completely obliterate stigma imprinted prior to their prehistoric collection, because the main late-phase change that occurs on flat surfaces prior to their collection is preserved in the hollows of remnant natural surfaces on archaeological tools. With the scanning electron microscope, five distinct morphologies representative of different environments have been characterised by establishing associations between the different forms and by plotting their regional distribution.     

Cyclic changes in the surface structure of the cervix from the ewe as revealed by scanning electron microscopy

Thirty parous ewes were divided into six groups and sacrificed on day 0 (first day of estrus), 1, 2, 10, 15 or 16 of the estrous cycle. The cervices were removed immediately and processed for examination with the scanning electron microscope. Observation of the tissues reveals that the surface of the cervix is highly convoluted, which results in the formation of numerous folds or crypts. Two forms of columnar epithelial cells, a ciliated and a non-ciliated cell with microvilli, line the luminal surface of the cerix in the day 10, luteal-phase ewes. However, on day 15, 2 days before estrus, the non-ciliated cells differentiate into two morphologically distinct types of secretory cells. One type forms when the apex of the non-ciliated cell dilates outward into the lumen of the cervix. Concurrent with apical enlargement, the microvilli are lost and the limiting cell membrane becomes smooth. The other type of cell is characterized by only a slight apical swelling. Consequently, remnants of microvilli along with secretory granules can be observed on the limiting membrane of this cell. Both cells release a particulate component, which is believed to be a precursor of mucus, into the lumen of the cerix. These particles undergo a series of morphological transformations to form a fibrillar layer, generally referred to as 'cervical mucus', that covers the epithelial surface at estrus. One to 2 days following the onset of estrus, the fibers become more closely assoicated with amorphous material that begins to coagulate, thereby revealing the underlying ciliated and non-ciliated cells that characterize the cervix of the luteal-phage ewe. The cyclical variation in secretory cells and factors that may influence that structural transformations which occur in mucus are discussed.     

External morphogenesis of the tardigrade Hypsibius dujardini as revealed by scanning electron microscopy

Tardigrada, commonly called water bears, is a taxon of microscopic panarthropods with five‐segmented bodies and four pairs of walking legs. Although tardigrades have been known to science for several centuries, questions remain regarding many aspects of their biology, such as embryogenesis. Herein, we used scanning electron microscopy to document the external changes that occur during embryonic development in the tardigrade Hypsibius dujardini (Eutardigrada, Parachela, Hypsibiidae). Our results show an accelerated development of external features, with approximately 30 hrs separating the point at which external structures first become recognizable and a fully formed embryo. All segments appear to arise simultaneously between ∼20 and 25 hrs of development, and no differences in the degree of development could be detected between the limb buds at any stage. Claws emerge shortly after the limb buds and are morphologically similar to those of adults. The origin of the claws is concurrent with that of the sclerotized parts of the mouth, suggesting that all cuticular structures arise simultaneously at ∼30 hrs. The mouth arises as an invagination in the terminal region of the head at ∼25 hrs, closes later in development, and opens again shortly before hatching. The anlagen of the peribuccal lobes arise as one dorsal and one ventral row, each consisting of three lobes, and later form a ring in the late embryo, whereas there is no indication of a labrum anlage at any point during development. Furthermore, we describe limited postembryonic development in the form of cuticular pores that are absent in juveniles but present in adults. This study represents the first scanning electron micrographs of tardigrade embryos, demonstrating the utility of this technique for studying embryogenesis in tardigrades. This work further adds an external morphological perspective to the developmental data already available for H. dujardini , facilitating future comparisons to related panarthropod taxa. J. Morphol. 278:563–573, 2017. © 2017 Wiley Periodicals, Inc.