The correlation of light and electron microscope observations

A technique is described to allow electron microscopic investigation of a specific feature of a section on a glass slide. A section on a glass slide (previously treated with a silicone release agent) is processed as required for light microscopy. The section is then impregnated with Araldite and cured with an epoxy resin block on top of it. The section and block are removed from the slide and viewed with a light microscope. The selected area for ultrastructural study remains under continuous observation while the block is trimmed. Semi-thin (1 μm) sections retain the original staining for light microscopy and ultra-thin sections are stained with heavy metals in the normal manner. We show how an inflammatory lesion in a large area of muscle in a case of polymyositis may be quickly located and studied at the ultrastructural level.     

Scanning electron microscopy of blood cells.

Scanning electron microscopy (SEM) is an invaluable tool for studying the surface morphology of isolated blood cells. We have used a simple preparative technique for SEM to study the erythrocytes from a case of Congenital Dyserythropoietic Anaemia type II and the leucocytes from healthy individuals or patients with leukaemia or lymphoma. This rapid and inexpensive technique for fixation and dehydration of blood cells was found to be equally reliable for obtaining micrographs of healthy or diseased leucocytes or erythrocytes.     

Scanning electron microscopy of uncoated human metaphase chromosomes.

Human metaphase chromosomes were processed with a 3% glutaraldehydetannic acid technique and examined in a scanning electron microscope at 20 kV either without added metal coating or with 2 nm of sputtered gold coating. Several substrates--aluminum mnium foil, silver mirror deposit and sputtered gold-provided good conductive backgrounds for chromosomal spreads. Silver mirror deposit was the best conductive substrate tested. This method should prove to be a useful tool for monitoring the three-dimensional morphology of mitotic chromosomes with the possibility of studying various banding techniques, chromosomal uncoiling and secondary constrictions currently being examined in chromosomal studies.     

Ultrastructure research in biology before the introduction of the electron microscope

Techniques used by the authors for preparing wood samples for the scanning electron microscope are detailed. Topics covered include: procedures for cutting, cleaning, dehydrating, mounting and storage of the specimens.     

Scanning electron microscope localization of zinc granules in the oyster ostrea angasi by backscattered electron imaging

A method for rapid localization of the zinc granules in oyster tissue using SEM has been developed. The results are discussed in terms of electron backscattering coefficients η for tissue, carbon support and embedding medium. Also, the application of the presented method for marine environment pollution detection and monitoring is recommended.     

Scanning reflection electron microscopy of surface topography by diffusely scattered electrons in the scanning electron microscope

In this paper, the technique of scanning reflection electron microscopy (SREM) by diffusely scattered electrons in the scanning electron microscope is described in detail. A qualitative account of the formation of image contrast in SREM is also described. It is assumed that, for grazing geometry, forward-scattered electrons reflect from regions close to the surface, following a few scattering events within the first few atomic layers, and lose very little energy in the process. The penetration depth of the primary electrons is very limited, resulting in strongly peaked envelopes of forward-scattered electrons. It is also assumed that a surface containing topographic features presents a range of tilt angles, resulting in different reflection coefficients. Tilt contrast results because each facet has a different scattering yield, which is dependent upon local surface inclination. Full details of the instrumentation designed for SREM are described, and to illustrate the technique, results recorded from an epitaxial GaAs on GaAs crystal, Pb₂(Zr,Ta)O₆ thin film on silicon, and SiO₂ amorphous film on silicon are presented.     

Capillary-tube units for individual block preparation in light and electron microscopy

Short lengths of 4-mm capillary tubing are fitted with teflon caps for processing individual blocks in preparation for light- and electron-microscopic observation. The processing units hold a nickel mesh grid to prevent tissue loss. Advantages include: (1) the ability to process very small blocks otherwise lost during processing; (2) separation of tissue from specifically mapped areas; (3) conservation of processing fluids and time. For routine tissue dehydration and infiltration, it is generally possible to process many tissue blocks collectively. However, instances frequently occur in which it is necessary to treat individual blocks separately. Examples include processing of single muscle fibres, and individual sensory regions where it is desirable to separate various segments of the sensory organ. This paper describes specially prepared capped capillary tubes for the purpose of individual block preparation. These containers are advantageous in conservation of fluids, ease of specimen transfer, and most importantly strict isolation of the individual blocks of tissue.     

Ultrastructural imaging of freeze-fractured plant cells in the scanning electron microscope.

The application of the freeze-fracture and cytoplasmic maceration technique in ultrastructural studies of plant cells is described. A major advantage of the technique is, that by extracting mobile cytoplasmic components from the freeze-fractured cells, surface relief is introduced and three-dimensional information is obtainable. The details of specimen preparation are described and the results obtained are reviewed. The use of chitosan embedding for very small or fragile specimens is described.     

A new method for comparative light and electron microscopic studies of individual cells,selected in the living state

A method is described which permits comparative light and electronmicroscopic studies of cell cultures, cell spreads or single selected cells which have been kept in the Plastic Film Dish (PFD). The PFD is a versatile large surface tissue culture chamber which, for electron microscopy, is mounted with a transparent FEP-Teflon film bottom. Cells are observed, selected and marked on the PFD-bottom with a high power inverted light microscope. The cells are fixed and dehydrated with a semi-automatic device while they are still in situ in the PFD. During the preparation steps for electron microscopy the topographical relationship between individual cells and between cells and cell support is accurately retained. After embedding and polymerization the Teflon film is easily peeled off the polymerized Epon, leaving a replica of the mark around the selected cell. This permits relocation of the selected cells for ultrathin sectioning in a plane plan-parallel to the original cell support. To enable orientated sectioning of selected cells in a plane perpendicular to the cell support, cells are tagged with Letraset-letters after original embedding and polymerization. Subsequently the re-embedded polymerized specimens are orientated in the microtome in a position which permits controlled thin sectioning of the tagged cells in the previously selected plane.     

Scanning electron microscopy of nerve fibers in human fetal cochlea

The cochleas of four human fetuses ranging 22–25 weeks gestation were studied by scanning electron microscopy (SEM) for the purpose of obtaining a better understanding of the nerve fiber arrangement in the human ear. After critical point drying, the specimens were dissected and the floor of the tunnel of Corti and the outer wall of Nuel's space were exposed for observation. Upper cochlear turns, especially the apical turn, seemed to be still immature. Observed nerve fibers were classified into three types:

• 1 Spiral fibers: Fibers traveling basalward and following the shape of the cochlea were found in both the tunnel of Corti and Nuel's space and believed to be the afferent nerves responsible for innervating the outer hair cells
• 2 Radial fibers: radiating outward from the osseous spiral lamina—one such radial fiber transversing high in the tunnel space (supposedly the efferent nerve servicing the outer hair cells), and another sort of radial fiber (found crossing the tunnel floor), the nature of which was uncertain.
• 3 Irregular fibers: Consisting of thin, randomly running fibers within the cochlea. The destination of these fibers was not determined, but possibly they represent transitory nerve branchings of afferent or more probably efferent nerves, which would later regress during maturation.

     

Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation

A potential strategy to enhance ligament healing by transplantation of mesenchymal stem cells (MSCs), which are demonstrated to differentiate into fibroblast-like cells in vitro, is presented. The objective of this study was to follow transplanted nucleated cells from bone marrow, which contain MSCs, in the healing medial collateral ligament (MCL) over time, and to examine their phenotype and survivability. It was hypothesized that MSCs in nucleated cells from bone marrow would differentiate into fibroblast-like cells in the healing ligament following adaptation to the environment. The transplantation model employed in this study eliminates the immune response to a donor by the recipient using a transgenic rat (donor), which does not produce foreign protein from transgenes, and its wild-type rat (recipient) in order to simulate autologous transplantation. The MCL of the wild-type rat was ruptured, where 1 x 10(6) nucleated cells of bone marrow from the transgenic rat were injected. The transgenes in transplanted nucleated cells were detected throughout the healing MCL for 28 days by in situ hybridization. At 3 days, many donor cells were evident in the injury site and fascial pocket, and some were found in the midsubstance. Morphologically, transplanted cells with elongated nuclei were found at the ruptured edge of the midsubstance and surface of the unruptured site after 3 days. At 28 days, these cells continued to survive in the healing MCL. Their shapes were similar to those of surrounding recipient MCL fibroblasts. Thus, transplanted cells might differentiate into fibroblasts. Therefore, it was demonstrated that there is a potential for nucleated cells from bone marrow to serve as a vehicle for therapeutic molecules as well as to be a source in enhancing healing of ligaments.     

Ionic liquid enables simple and rapid sample preparation of human culturing cells for scanning electron microscope analysis

Ionic liquid is a kind of salt that stays in a molten state even at room temperature. It does not vaporize at all in vacuum and facilitates electrical conductivity to the sample surfaces for observations with a scanning electron microscope (SEM). In this study, we used an ionic liquid in SEM for the first time to observe fixed human culture cells. The condition for the cell culture using wrapping sheets and SEM settings were varied to elucidate the optimized protocol. Compared to samples prepared by the conventional way, the ionic liquid‐treatment of samples gave SEM images of the cellular ultra structures in more detail, enabling observation of microvilli that made bridges between separated cells. In addition, the ionic liquid treatment is less time consuming as well as less laborious compared with the conventional way that includes dehydration, drying, and conductivity treatments. Totally, we concluded the ionic liquid is a useful reagent for SEM sample preparation. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Use of light and scanning electron microscope techniques to monitor microstructural changes in aluminium-based metal matrix composites

Microstructural developments in discontinuously reinforced aluminium composites have been monitored during a variety of thermo-mechanical treatments. The material, made by powder blending or casting, followed by extrusion, contains particulate or short-fibre alumina in a matrix of commercial purity aluminium. Attention is centred on the distribution of reinforcement, the fine alumina particles derived from the prior oxide layers on the aluminium powder, and the effects that these inclusions have on cavitation, recovery and recrystallization processes. Optical metallography, by viewing anodized sections in polarized light, is used to reveal grain and coarse subgrain structures. Scanning electron microscopy has also been employed to reveal fine oxide stringers, cavities and (in backscattered mode) fine subgrain structures. The emphasis in this paper is on the type of information that can be obtained for these materials from careful application of standard microscopy techniques.     

Scanning electron microscope for in situ study of crystallization of Ge2Sb2Te5 in phase-change memory

By introducing electrical connections into the chamber of a scanning electron microscope (SEM) via its holder assembly, it has become feasible to in situ observe and electrically characterize electronic devices. The in situ SEM was applied to investigate electric-pulse-induced behavior of Ge(2)Sb(2)Te(5) in a lateral phase-change memory cell. Randomly distributed nuclei with sizes from 20 to 80 nm were initiated at a low voltage pulse. Initially, grain growth depended strongly on pulse amplitude at around 60.3 nm/V and then a weak pulse amplitude dependence was observed at around 13.5 nm/V. Device resistance during crystallization dropped by two to three orders of magnitude with two falling steps, which probably resulted from amorphous to face-centered-cubic and subsequently to hexagonal transitions, respectively.     

High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat

We analyzed the effect of glucocorticoid on bone regeneration after bone marrow ablation in tibiae of 8-week-old rats. Methylprednisolone sodium succinate (MPSS) was injected intramuscularly at a dose of 100 mg/kg/day for 3 days. Tibiae on days 1, 3, 5, 7, 10, 12, and 14 after ablation were subjected to tartrate-resistant acid phosphatase staining, immunohistochemistry, in situ hybridization, and transmission electron microscopy (TEM), and measurement of the volume of newly-formed bone and the osteoclast number. MPSS significantly decreased the newly-formed bone volume on day 7, and immature bone still remained on day 10 in the MPSS-treated group. The volume of this bone was significantly higher than that in the control group. However, there were no differences between the groups in the osteoclast number, the expression of mRNAs for osteoblast differentiation markers, and alkaline phosphatase and cathepsin K judged by immunohistochemistry. TEM findings showed no difference in the form of osteoblasts, whereas osteoclasts in the MPSS-treated group had less developed ruffled borders, compared to those in the control group. These results suggest that MPSS treatment affects neither the differentiation nor the shape of osteoblasts, and does not change the osteoclast number or the cathepsin K level. However, high dose MPSS inhibits both bone formation and resorption during bone regeneration after rat tibial bone marrow ablation, and inhibits ruffled border formation in osteoclasts. These data will be useful to develop bone regenerative therapies for bone diseases due to high dose steroid administration.