Resin-embedded Semithin Sections Research Articles

Imaging the Kidney with an Unconventional Scanning Electron Microscopy Technique: Analysis of the Subpodocyte Space in Diabetic Mice.

Transmission electron microscopy (TEM) remains the gold standard for renal histopathological diagnoses, given its higher resolving power, compared with light microscopy. However, it imposes several limitations on pathologists, including longer sample preparation time and a small observation area. To overcome these, we introduced a scanning electron microscopy (SEM) technique for imaging resin-embedded semi-thin sections of renal tissue. We developed a rapid tissue preparation protocol for experimental models and human biopsies which, alongside SEM digital imaging acquisition of secondary electrons (SE–SEM), enables fast electron microscopy examination, with a resolution similar to that achieved by TEM. We used this unconventional SEM imaging approach to investigate the subpodocyte space (SPS) in BTBR ob/ob mice with type 2 diabetes. Analysis of semi-thin sections with secondary electrons revealed that the SPS had expanded in volume and covered large areas of the glomerular basement membrane, forming wide spaces between the podocyte body and the underlying filtering membrane. Our results show that SE–SEM is a valuable tool for imaging the kidney at the ultrastructural level, filling the magnification gap between light microscopy and TEM, and reveal that in diabetic mice, the SPS is larger than in normal controls, which is associated with podocyte damage and impaired kidney function.

Applications of Scanning Electron Microscopy Using Secondary and Backscattered Electron Signals in Neural Structure.

Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography (i.e., serial section SEM). This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods.

Efficient fluorescence recovery using antifade reagents in correlative light and electron microscopy.

Correlative light and electron microscopy (CLEM) enables ultrastructural-level analysis of fluorescence-labeled proteins by combining images obtained from both fluorescence and electron microscopies. A technical challenge with the CLEM method is the effective detection of fluorescence from samples embedded in resins, which generally cause fluorescence decay. To overcome this issue, we developed a method for fluorescence recovery of green fluorescent protein (GFP) in resin-embedded semi-thin sections using commercially available antifade reagents. By applying this method, we successfully obtained CLEM images using field-emission scanning electron microscopy with moderately enhanced GFP signals, demonstrating the efficacy of this simple fluorescence recovery method.

Immunolocalization to Study ARGONAUTE Proteins in Developing Ovules of the Brassicaceae.

Determining the in situ pattern of protein expression is crucial to accurately establish regulatory function and mode of action of any plant developmental program. Here, we describe two immunolocalization procedures that are consistently used to determine subcellular localization of ARGONAUTE proteins in the ovule of the Brassicaceae. The first is performed in resin-embedded semi-thin sections of developing ovules that can be observed under bright-field microscopy. The second is based in polyacrylamide immersion of complete (whole-mounted) gynoecia or ovules that are observed under confocal microscopy. Both procedures have been successfully performed to localize proteins involved in RNA-directed DNA methylation during the development of the anatropous bitegmic ovule in Arabidopsis, Brassica, or Boechera species.

Cyanoacrylate glue as an alternative mounting medium for resin-embedded semithin sections

Commercially available generic Superglue (cyanoacrylate glue) can be used as an alternative mounting medium for stained resin-embedded semithin sections. It is colourless and contains a volatile, quick-setting solvent that produces permanent mounts of semithin sections for immediate inspection under the light microscope. Here, we compare the use of cyanoacrylate glue for mounting semithin sections with classical dibutyl phthalate xylene (DPX) in terms of practical usefulness, effectiveness and the quality of the final microscopic image.

A simple protocol for paraffin‐embedded myelin sheath staining with osmium tetroxide for light microscope observation

Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.

Delayed postnatal settlement of cerebellar Purkinje cells in vermal lobules VI and VII of the mouse

The postnatal development of the ganglionic (Purkinje) layer was studied in the mouse cerebellum from P0 to young adulthood with special emphasis to vermal lobules VI-VII (oculomotor vermis) in the mouse. In order to visualize Purkinje cells (PCs), toluidine blue staining of resin-embedded semithin sections and calbindin immunohistochemistry were utilized. The number of PCs in the whole cerebellum was 199,080+/-2966 at postnatal day eight (P8), 222,000+/-2979 at P20 and nearly the same, 225,800+/-7549 in young adults; i.e., there was an approximately 13.4% increase of PCs between P8 and adults. The number of PC somata aligned into a rostrocaudal stripe along the developing ganglionic layer increased by about 24% in vermal cerebellar lobule III but much more markedly (i.e., by 49%) in VI+VII between P6 and young adulthood. Between P6 and P16, the increase of the number of PCs in the ganglionic layer of lobules VI and VII resulted in the (delayed) completion of PC layer, caused by the (late) alignment of rostrocaudally dispersed PCs, although late postnatal migration of a smaller population of these cells cannot be excluded either. It is concluded that the oculomotor vermis belongs to the latest developing cerebellar cortical structures, which could be the reason for its frequent involvement in developmentally related disturbances and disorders.

Microcanalization in the epididymis to overcome ductal obstruction caused by chronic exposure to chromium - a study in the mature bonnet monkey (Macaca radiata Geoffroy).

In order to apprehend the toxic effects of chromium, an occupational/environmental pollutant, on the epididymis, adult bonnet monkeys were exposed to chromium (VI) in their drinking water at concentrations of 100, 200 and 400 p.p.m. for a chronic period of 180 days. At the end of the experimental period, testicles and segments of epididymis from control and treated monkeys were subjected to light microscopic (resin-embedded semi-thin sections) and transmission electron microscopic analyses. Among the various changes undergone by the epididymal epithelium, the present paper describes the origin of two different kinds of microcanals, probably caused by ductal obstruction. The first type of microcanal, which appears to provide passage for spermatozoa to bypass the obstructed main duct, is comparable with the one already reported in carbendazim-treated efferent ductules of the rat. The second type of microcanal, which is novel, consisted of a lumen in the epithelium enclosed by four to five cells, which are either modified basal cells, principal cells or a hitherto unknown cell type. This novel type of microcanal is suggested to be a device to entrap the spermatozoa which reach the core of the epithelium and may be a mechanism to prevent extravasation of sperm so as to avoid an autoimmune response of spermatic granuloma formation. Thus, the present study has shown that chronic exposure to chromium (VI) through drinking water can produce pathological manifestations in the epididymal epithelium but the epididymis, being a versatile organ, is capable of overcoming such adverse situations through novel devices.

Computer-aided morphometric analysis of the developing concentric structure of the human fetal intestinal tube.

The Image-Pro Plus 3.0 morphometric program was used to study the region-specific organization of the human fetal intestine across the radial axis of the gut at weeks 12 and 18 of gestation. The thicknesses of the epithelium, the submucosa, the muscular layers and the myenteric ganglia were measured in resin-embedded semithin sections. Statistical analysis of the collected data was performed by using the two-way ANOVA, the SNK test and the Pearson correlation. The structural changes relating to the gut morphogenesis within this developmental period were followed both light and electron microscopically. The various tissues forming the radial axis of the intestinal tube exhibited different trends concerning their individual development. The thickness of the epithelium did not change in the fetal period investigated, although the epithelial surface displayed characteristic ultrastructural changes. The thickness of the submucosal layer increased significantly, but with different dynamics along the longitudinal axis, whereas the increases in size of the muscular layers and the myenteric ganglia did not differ significantly along the longitudinal axis of the embryonic intestine. The Pearson correlation revealed a significant correlation between the development of the circular muscle layer and that of the myenteric plexus along the whole length of the intestinal tube. The epithelium, the submucosa and the longitudinal muscle layers developed independently between weeks 12 and 18 of gestation.

Pale vacuolated epithelial cells in epididymis of aflatoxin-treated mice.

The responses of the mouse epididymal epithelium to subchronic doses of aflatoxin B1 were investigated in a histological study. Either few and large or small and profuse vacuoles containing an amorphous to dense periodic acid-Schiff-positive material were observed in the epithelium of all the segments of the epididymis. Resin-embedded semi-thin sections and transmission electron microscopy indicated that these vacuoles were intracellular. The cells that contained these vacuoles were quite different in organization and electron density from the cell types already established in the epididymal epithelium and are designated as pale vacuolated epithelial cells. Owing to aflatoxin B1 toxicity, the apical membrane of some of the principal cells, either individually or in groups, disintegrated so that the principal cells released their contents into the lumen of the duct through development of a 'fistula'. Spermatozoa from the ductal lumen entered the principal cell fistula and reached the basal lamina. If extravasation of the spermatozoa via this route occurred, it would bring about an autoimmune response, leading to the formation of spermatic granulomas and the generation of anti-sperm antibodies. Extravasation of spermatozoa seems to be offset by the underlying basal cell, which is presumed to develop into a pale vacuolated epithelial cell to enclose the disintegrating principal cells and the spermatozoa arriving at the principal cell. Thus, the development of pale vacuolated epithelial cells may be a protective device preventing an autoimmune response to sperm antigens in the context of toxicant-induced degeneration of the principal cells of the epididymal epithelium.

Pale vacuolated epithelial cells in epididymis of aflatoxin-treated mice

The responses of the mouse epididymal epithelium to subchronic doses of aflatoxin B1 were investigated in a histological study. Either few and large or small and profuse vacuoles containing an amorphous to dense periodic acid-Schiff-positive material were observed in the epithelium of all the segments of the epididymis. Resin-embedded semi-thin sections and transmission electron microscopy indicated that these vacuoles were intracellular. The cells that contained these vacuoles were quite different in organization and electron density from the cell types already established in the epididymal epithelium and are designated as pale vacuolated epithelial cells. Owing to aflatoxin B1 toxicity, the apical membrane of some of the principal cells, either individually or in groups, disintegrated so that the principal cells released their contents into the lumen of the duct through development of a 'fistula'. Spermatozoa from the ductal lumen entered the principal cell fistula and reached the basal lamina. If extravasation of the spermatozoa via this route occurred, it would bring about an autoimmune response, leading to the formation of spermatic granulomas and the generation of anti-sperm antibodies. Extravasation of spermatozoa seems to be offset by the underlying basal cell, which is presumed to develop into a pale vacuolated epithelial cell to enclose the disintegrating principal cells and the spermatozoa arriving at the principal cell. Thus, the development of pale vacuolated epithelial cells may be a protective device preventing an autoimmune response to sperm antigens in the context of toxicant-induced degeneration of the principal cells of the epididymal epithelium.

A method for collecting semithin epoxy serial sections for light microscopy and 3-D reconstruction

In the three-dimensional reconstruction of neuronal structure, it is imperative that ribbons of semithin or ultrathin sections be obtained. Resin-embedded semithin sections display better structural details than paraffin-embedded sections. The cutting and collecting of long ribbons of resin-embedded semithin sections using a microtome, requires the use of large troughs on glass knives. A simple plastic trough has been described which facilitates the collection of ribbons directly onto a coverslip. As the ribbons are formed, they are floated on a coverslip. The water in the trough is slowly drained through a tubing which is attached to a syringe. The ribbons settle on the coverslip, which is easily removed and placed on a hotplate to dry the sections.