Tridimensional ultrastructure of perfusion fixed gastrointestinal epithelial cells by high resolution scanning electron microscopy

Improvements in the design of modern scanning electron microscopes (SEM) and new methods of specimen preparation incorporating chemical removal of the cytosol and cytoskeleton, now make it possible to view cells and their organelles in three dimensions (3D) at high magnification. In this experiment, high resolution SEM (HRSEM) utilizing new methods of tissue preparation was used to study the intracellular structures of the mouse ileum. In addition, in vivo intestinal perfusion was used to further enhance cellular preservation. Using these modifications it was possible to visualize, in 3D, the fine structure of intestinal epithelial cells and intracellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi complex, as well as microvilli and cell membrane. Whole mitochondria appeared as irregularly shaped organelles which contained tubular cristae. Plate-like cristae were not observed. The brush border was found to be a closely packed array of cylindrical projections. The extensive folding and structural intricacy of lateral cell membranes between absorptive cells could only be appreciated by viewing this tissue with 3D HRSEM. The use of HRSEM to study 3D ultrastructure of cells and their organelles will improve our understanding of the structure-function relationships in both the healthy and diseased gastrointestinal tract.     

Ultrastructure of the seminiferous epithelium and intertubular tissue of the human testis

The ultrastructural features of the human testis are reviewed with emphasis upon the process of spermatogenesis and the cytology of the Leydig cells. The seminiferous epithelium is structurally partitioned by the Sertoli cells into basal and adluminal compartments via the specialized tight junctions between the Sertoli cells. Spermatogonia reside in the basal compartment, and, via a series of cell divisions, produce the primary spermatocytes, which at the commencement of their development move into the adluminal compartment, and thus the lengthy process of meiotic maturation is initiated. The fine structure of primary spermatocytes is described together with the complex transformation of the spermatids into spermatozoa during the process of spermiogenesis. Earlier studies of the organization of the human seminiferous epithelium showed that germ cells at different developmental stages formed identifiable collections termed cell associations or stages, but since several stages were seen in a single tubule cross-section, this gave the impression of an extremely irregular pattern of spermatogenic development. When the topographic arrangement of germ cells was re-examined with the aid of computer modelling, a highly ordered distribution was revealed, conforming to a helical pattern based on the geometry of spirals. Thus spermatogenesis in the human testis is subjected to a precise regulation in keeping with the ordered arrangement of the germ cells seen in other mammalian species. The intertubular tissue of the human testis is composed of loose connective tissue containing blood vessels, occasional lymph capillaries, macro-phages, mast cells, and the Leydig cells which occur either as single cells or form small clusters. The Leydig cell cytoplasm contains an abundant supply of smooth endoplasmic reticulum and mitochondria with tubular cristae, both features being characteristic of steroidogenic cells. Human Leydig cells contain large Reinke crystalloids of variable size and number, but their function remains obscure. The frequent occurrence of paracrystalline inclusions within the cytoplasm of the human Leydig cell suggests that these elements are precursors of the Reinke crystalloids.     

Serial section reconstruction using a computer graphics system: Applications to intracellular structures in yeast cells and to the periodontal structure of dogs' teeth

A computer graphics system for reconstruction from serial section micrographs was applied to intracellular details of a yeast target cell (Saccharomyces cerevisiae cell) induced by the α factor mating pheromone and was also applied to a periodontal structure of a dog tooth moved orthodontically. In the former, intracellular organelles and a distribution of vesicles could be clearly observed through the cell membrane using the transparent display method in which the smoothing of the reconstructed outer cell membrane surface by computer processing was applied to the transparent display. In the latter case, by cutting through a reconstructed dog tooth and its periodontal tissues, labiolingual and mesiodistal cut surfaces of the tooth and of adjacent alveolar bone could be observed with fine details (232 sections were used).     

Structural and functional alterations of cellular components as revealed by electron microscopy

Scanning (SEM) and transmission electron microscopy (TEM) are two fundamental microscopic techniques widely applied in biological research for the study of ultrastructural cell components. With these methods, especially TEM, it is possible to detect and quantify the morphological and ultrastructural parameters of intracellular organelles (mitochondria, Golgi apparatus, lysosomes, peroxisomes, endosomes, endoplasmic reticulum, cytoskeleton, nucleus, etc.) in normal and pathological conditions. The study of intracellular vesicle compartmentalization is raising even more interest in the light of the importance of intracellular localization of mediators of the signaling in eliciting different biological responses. The study of the morphology of some intracellular organelles can supply information on the bio‐energetic status of the cells. TEM has also a pivotal role in the determination of different types of programmed cell death. In fact, the visualization of autophagosomes and autophagolysosomes is essential to determine the occurrence of autophagy (and also to discriminate micro‐autophagy from macro‐autophagy), while the presence of fragmented nuclei and surface blebbing is characteristic of apoptosis. SEM is particularly useful for the study of the morphological features of the cells and, therefore, can shed light, for instance, on cell–cell interactions. After a brief introduction on the basic principles of the main electron microscopy methods, the article describes some cell components with the aim to demonstrate the huge role of the ultrastructural analysis played in the knowledge of the relationship between function and structure of the biological objects. Microsc. Res. Tech., 76:1057–1069, 2013. © 2013 Wiley Periodicals, Inc.     

High resolution SE-I SEM study of enamel crystal morphology

Until recently high resolution TEM was the only imaging mode capable of probing the atomic lattice structure of crystals composing tooth enamel. Studies designed to determine the polyhedral shape of normal enamel crystals and initiation of carious lesions in enamel crystals were hampered and limited by interpretation of two-dimensional TEM images from thin section and freeze fracture replica specimens lacking depth of field. The newly developed SE-I signal mode for SEM (SE-I/SE-II ratio) can produce images of enamel crystals approaching beam diameter dimensions (0.7–2.0 nm), rivaling the resolution of the TEM technique and generating topographic contrasts for three dimensional imaging at very high magnification (≈?1,000,000 X). Ultrathin chromium (Cr) films generate enriched high resolution SE-I contrasts of enamel crystal surfaces and when imaged using an immersion lens field emission SEM operated at high voltage (20–30 KeV) produce unsurpassed topographic contrasts. Since the grain size of Cr is below the resolution of any SEM and is ultrathin (≈?1 nm), then SE-I images can provide a more accurate representation of enamel crystal structure than TEM methodologies. Our SE-I SEM observations of normal human enamel crystals reveal fractured spicules which contain angled flat surfaces delineated by a prominent 2 nm wide SE-I edge brightness contrast. Although microscopic observations often show crystals which are hexagonal in cross-section, in both SEM and TEM many other growth habits, including rectangular or irregular crystals (30–40 nm in width) which contain “notches,” are also observed. More detailed morphological studies are therefore required to determine the most likely habit planes and their relevance to the function of the enamel crystals. The granular appearing fine structural contrast imposed onto <100> lattice planes of sectioned enamel in TEM micrographs is also resolved with topographic contrasts in SE-I micrographs. These granules probably represent one or both of the enamel protein classes.     

Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM)

We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.     

The histomorphological structure of the male reproductive system of maize leaf weevil Tanymecus dilaticollis Gyllenhal, 1834 (Coleoptera: Curculionidae)

The histomorphology of the reproductive system and the germ cells has been useful to establish phylogenetic relationships in many insects. However, these elements remain little known in the Curculionidae. In this study, histomorphological structure of the male reproductive system of Tanymecus dilaticollis, which is economically important, is described, illustrated using stereomicroscopy, light microscopy, and scanning electron microscopy techniques, and discussed in relation to other Coleoptera species. Results showed that distinctive features of the male reproductive system of T. dilaticollis consist of a pair of yellowish testes, a pair of seminal vesicles, a pair of vasa deferentia, an ejaculatory duct, accessory glands, prostate glands, and aedeagus. Each testis is subdivided into two testicular follicles, enclosed by a peritoneal sheath. Each follicle of the mature testes is full sperm cysts with germ cells at various stages development of spermatogenesis. The testes have four types of germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa). They are occupied by the growth zone containing spermatogonia and spermatocytes, the maturation zone containing spermatids, while differentiation zone containing spermatozoa. There is a seminal vesicle at the center of each testis. Most mature sperms are stored in the seminal vesicle. Each testis is attached to the vas deferens by a stalk‐like seminal vesicle. In the distal part, vasa deferentia fuse with the ejaculatory duct. It is linked to the aedeagus. The provided results will contribute to the understanding of the reproductive cell biology of Curculionidae.     

Aberrant distribution of junctional complex components in retinoic acid receptor alpha‐deficient mice

Retinoic acid receptor alpha (RARα)‐deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell–cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli‐cell barrier in the tubules of RARα‐deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)‐responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO‐1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin‐40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RARα‐deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RARα‐deficient testes may correlate with a disrupted cyclic expression of RA‐responsive structural components, including vimentin, a downregulation of connexin‐40 in spermatogenic cells, and delayed assembly of ZO‐1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

The use of geometrical models as a stereological aid exemplified by the solidification geometry of ferrous metals

In the present paper, model geometry is used to describe the three-dimensional nature of some structural constituents of ferrous metals, such as cells, grains, dendrites, networks etc. in the course of their solidification process. Based on micrographs of eutectic cells, highly magnified model structures were built. By cutting the models, all the features found in real micrographs (cell boundaries, groupings, phosphide figures etc.) could be simulated. Using the known geometrical properties of the models, parameters such as the porosity due to phosphides, can be calculated; this allowed precautionary casting techniques to be derived. Further, conclusions could be drawn about processes occurring during structure formation, i.e. during cooling and solidification of the metal. A geometrical derivation of various equations for internal surfaces, hydraulic diameters during the last moments of crystallization etc., and the technological applications of this knowledge are possible. As structural constituents of metals are sometimes complicated bodies, our more intuitive geometrical approach using models often provides more reliable information about their appearance than very elegant but abstract arithmetical methods.     

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.     

Discussions on the multiple-input maximum a-posteriori wave-function restoration method in high-resolution electron microscopy

The multiple-input maximum a-posteriori (MIMAP) wave-function restoration method in high-resolution electron microscopy proposed by Kirkland (1984) is further discussed. A modified MIMAP criterion that considers likely correlations between individual micrographs in a series of micrographs taken at different imaging conditions is presented. By performing wave-function restoration for a multislice method-simulated defocus series of MgO[110], the convergent properties of this modified MIMAP method are also described.     

Seasonal ultrastructural changes in apocrine gland cells in back skin of male brown bears (Ursus arctos)

Oily secretions from the back skin are involved in the marking behavior of male brown bears (Ursus arctos), and apocrine glands in back skin are activated during the breeding season. Here, we investigated seasonal changes in the intracellular organelles of apocrine gland cells in the back skin of male brown bears using transmission electron microscopy (TEM) and osmium‐maceration scanning electron microscopy (OM‐SEM). The morphological features of mitochondria and intracellular granules, and secretory mechanisms obviously differed between breeding and non‐breeding seasons. The TEM findings showed that contents of low‐density granules were released into the glandular lumen by frequent exocytosis, and sausage‐shaped mitochondria were located in the perinuclear region during the non‐breeding season. In contrast, high‐density granules appeared in the apical region and in projections during the breeding season, and swollen mitochondria and lysosome‐like organelles separating into high‐density granules were located in the perinuclear region. The OM‐SEM findings revealed swollen mitochondria with only a few partially developed cristae, and small mitochondria with cristae shaped like those in swollen mitochondria in the apical regions during the breeding season. These findings indicated that the small mitochondria corresponded to the high‐density granules identified by TEM. These findings suggested that mitochondria in apocrine gland cells swell, degenerate, fracture into small pieces, and are finally released by apocrine secretions during the breeding season. Small mitochondria released in this secretory manner might function as the source of chemical signals in the oily secretions of brown bears during the breeding season.     

Noise reduction in high-magnification micrographs by soft focus printing and digital image processing

Soft focus printing of micrographs, using screens of opaque material with 10–15 lines/cm, reduced contrast due to noise but preserved all other contrasts and all structural details. Appropriate screens generated diffraction fringes of a spacing similar to the distance of the noise structures. The superimposition of fringes and image produced a diffusion effect which reduced only the contrast of the noise background. Blurring, screening or fogging were not effective or suitable for photographic micrograph processing. Soft focus printing improved recognition of weak high magnification contrasts in SEM micrographs of uncoated, gold decorated, and chromium coated cell surfaces of glomerular endothelium in mouse kidney slices. Effects similar to the optical diffusion were obtained by digital image processing using weighted low pass filters.     

Histology and ultrastructure of the testis and vas deferens in Pseudochorthippus parallelus parallelus (Orthoptera,Acrididae)

Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) is a widespread species in Europe, and also it is localized in some regions in Turkey such as Bursa, Eski?ehir, Ankara, Bolu, Düzce, and Çank?r?. The features of the reproductive organs such as the numbers and shapes of testes and follicles can be used as taxonomical characters. For this purpose, the ultrastructural and histological features of testis and vas deferens in P. parallelus parallelus were examined with using light microscope, scanning electron microscope, and transmission electron microscope. The mature P. parallelus parallelus has two conjugated testes produce spermatozoa. Each testis is composed of numerous testis follicles in which different stages of spermatogenesis and spermiogenesis develop. First, spermatocytes are formed by the mitosis division of the germ cells at the distal end of the follicles. Then, spermatocytes form spermatids by meiosis division in the middle region of the follicles. Finally, spermatids are differentiated to spermatozoa at the proximal region of the follicles. After maturation of the spermatozoa, sperm tails come together as the sperm bundles called as spermatodesm. Each follicle is connected to vas deferens via vas efferens to discharging spermatozoa. In spite of some differences, the testes and the vas deferens in P. parallelus parallelus are highly similar to the those of other species, especially Orthopteran species.     

Imaging of granular high-Tc thin films using a scanning tunnelling microscope with large scan range

STM images are presented in the micron scale, taken in UHV with a single-tube scanner STM with SEM control of the tip position. For calibration, a carbon grid was used from which the coupling of x and y scan axes has been determined as well as the piezo-sensitivity factors. Images taken of a YBa₂Cu₃O_7-δ, film display the granular structure of the crystallites formed during the post-evaporation annealing. A direct comparison of STM scans with SEM micrographs was made which demonstrates the complementary information obtained by the two methods. The obtained resolution is compared to the tip shape which becomes crucial for the imaging of corrugated surfaces on the micron scale. With a simple geometric model, an attempt has been made to reconstruct the surface topography from STM scans based on the knowledge of the tip shape.     

Scanning electron and light microscopy of foliar epidermal characters: A tool for plant taxonomists in the identification of grasses

SEM and light microscopy are of special interest for biologist to observe various features of the living bodies. In the current study we observed the foliar epidermal micro‐morphological characters of 44 grass species using SEM and Light microscopy to assess their taxonomic utility for taxonomists in the identification process. The aim of this study is to use the foliar epidermal structural variations in both upper and lower surfaces for identification of grasses. Significant diversity was observed in both qualitative and quantitative characters using SEM and Light microscopy. Variations were observed in stomatal number, size, guard cells shape, silica bodies, macro‐hairs, micro‐hairs, epidermal cell number, subsidiary cells, prickles, hooks, papillae, and short and long cells. A taxonomic key is prepared using these variations for the identification of grass species. Based on these findings, SEM and Light microscopy of foliar epidermal features can be of special interest for taxonomists in the identification of complex grass taxa.     

Proceedings of the eleventh annual meeting of the arizona society for electron microscopy and microbeam analysis held in tucson,arizona, february 7–8, 1990

The ultrastructure of the progressive testicular involution with advancing age in men is reviewed. There is no definite age at which testicular involution begins, and the onset and severity of testicular lesions are subjected to pronounced individual variations. Hormone studies also indicate great individual variations, and subtle changes in both the testis and the pituitary develop progressively with age. Testicular size, sperm quality, and numbers of all germ cell types, Sertoli cells, and Ley dig cells decrease with age. The volume occupied by the seminiferous tubules decreases, whereas that occupied by the testicular interstitium remains constant. The most frequent histological pattern of the aging testis is a mosaic of different seminiferous tubule lesions, varying from tubules with complete, although reduced, spermatogenesis, to completely sclerosed tubules. The tubules with complete spermatogenesis may show numerous morphological abnormalities in the germ cells, including multinucleation. Abnormal germ cells degenerate causing Sertoli cell vacuolation. These vacuoles correspond to dilations of the extracellular spaces resulting from the premature exfoliation of germ cells. Degenerating cells that are phagocytosed by the Sertoli cells give rise to an accumulation of lipid droplets in the Sertoli cell cytoplasm. The loss of germ cells begins with the spermatids, but progressively affects the earlier germ cell types, and tubules with maturation arrest at the level of the spermatocytes or spermatogonia are observed. The Sertoli cells show morphological abnormalities such as dedifferentiation, mitochondrial metaplasia, and multinucleation. Germ cell loss is associated with thickening of the tunica propria. When all seminiferous epithelial cells have disappeared, only an intensely collagenized tunica propria with myoid cells remains (sclerosed tubules). The Ley dig cells progressively dedifferentiate with a decrease in the quantity of both smooth endoplasmic reticulum and mitochondria, together with an accumulation of lipid droplets, crystalline inclusions, and residual bodies, and formation of multinucleate cells. The development of tubular involution with age is similar to that observed after exprimental ischemia, suggesting that vascular lesions may play an important role in age-related testicular atrophy.     

A method of microscopic evaluation of effects of demineralizers on complex carbohydrates of rat tissues

A technique is described in which the scanning electron microscope (SEM) has been used to study the internal cellular organization of the plant organs and the interaction of the plant with the environment. Transverse surfaces were revealed by fracturing quick frozen specimens and freeze-drying the fragments. The greater depth of focus of the SEM compared with the optical microscope enabled the production of clear, realistic micrographs with a three-dimensional appearance.     

Preparation of cultured cells for SEM: air drying from organic solvents.

Air evaporation from organic solvents of differing polarities and surface free energies was used in the preparation of cultured murine peritoneal macrophages for scanning electron microscopy (SEM). The surface structural features of these cells were compared to the surfaces of similar cells prepared by the critical-point procedure. In general, all organic solvents produced a marked collapse of cell structure resulting in an increase in surface irregularity. Fracture surfaces and sharply defined contours including numerous flaps and ridges were characteristic of the non-polar solvent dehydrated samples. Polar solvent dehydrated samples, including those dried from solvents of low surface free energy, exhibited a secondary flowing and settling of the cell membrane. Primary collapse was apparent but cell contours were smoothed and rounded. Overall cell shape and cell-to-cell relationships were retained regardless of solvent type. It is suggested that solvent evaporation may prove useful in certain cases, though investigators are advised to use caution when interpreting the results obtained by such procedures.     

Video-rate confocal reflection microscopy of neoplastic cells: Rate of intracellular movement and peripheral motility characteristic of neoplastic cell line (RSK4) with high degree of growth independence in vitro

Video-rate laser confocal interference reflection microscopy was used to demonstrate rapid motion of intracellular organelles and features at the cell periphery in a fully transformed neoplastic cell line, RSK4, and in four other neoplastic cell populations. In the RSK4 cells, vibrational and trafficking movements of intracellular particles at a rate greater than 25 Hz and ranging down to 5 Hz were recorded. Rapidly moving processes changed to ruffles, then microspikes, and previously undetectable ephemeral intercellular contacts were seen. Dynamic cyclical changes were revealed in the sizes of the podosomal close contacts of the transformed cells. The visibility of such features and the temporal and spatial resolution are improved over earlier methods. The fact that fast cellular and intracellular movements can be detected with this microscopic technique offers new possibilities in attempting to recognise differences between unimpaired living cells, and it may prove useful in the identification of malignant cells.