Contrast and quantitation in uniform regions of thin sections using transmission electron microscopy

A direct approach to quantitative measurements of uniform regions in thin sections is described. Accelerating voltages around 80 kV and objective aperture angles of about 9·3 mrad will provide conditions where contrast is directly proportional to specimen mass thickness. An extensive treatment of electron scattering in Formvar films for wide ranges of electron microscopic operating conditions is summarized in a simple, empirical equation. The extent to which Formvar results may be generalized to other materials, both embedding media and structures within the thin section, is treated. Using these results, precise measurements of local section thickness and of specimen density and/or dry mass of regions which penetrate the entire section thickness are possible, with the accuracy dependent upon irradiation effects and specimen makeup.     

An easy and versatile embedding method for transverse sections

In several research areas, transverse sections are indispensable for studying structural aspects of specimens. However, the oriented embedding of small cylindrical samples can become problematic, especially when transverse sections at right angles to the main axis of the object are desired. Here, we describe an easy and low‐cost technique for oriented embedding of small (? < 500 µm) as well as of larger specimens (? > 500 µm). The usefulness of the technique is demonstrated for roots and stamens of Arabidopsis thaliana and for adventitious roots of Asplenium demerkense, as examples of small and larger cylindrical samples, respectively. Furthermore, several types of resin (glycol methacrylate, epoxy and acrylic resins) were successfully tested, showing the applicability of the technique for light and electron microscopy and for immunolocalizations. In conclusion, the principle of the technique can be extended to several resins and a wide variety of specimen types, such as stems, leaves and textile fibres. The originality of the technique lies in its simplicity combined with its high efficiency to produce well‐oriented transverse sections.     

Effect of counterface material for abrasive wear of Cercidiphyllum japonicum wood on three-body abrasion

A three-body abrasion test with a loose abrasive grain scattered on a variety of plastic counterface materials is conducted for Cercidiphyllum japonicum wood (katsura wood). The effect of the counterface material in rubbing with the katsura wood is investigated. The results show that a peak wear coefficient exists for the axial, tangential and radial sections of the katsura wood specimen when rubbed with a counterface material. The peak in the wear coefficient is also recognized in the plastic specimen experiments. The peaks in three-body abrasion experiments for both the katsura wood and plastic specimens are closely related to the variable of material yield stress. The peak on katsura wood specimen occurs when the yield stress of the counterface material is approximately twice as large as that of katsura wood, and the peak on the plastic specimen occurs when the yield stress of the counterface material is approximately the same as that of the specimen. The difference in the results between the katsura wood and plastic material could appear to be due to the change in embedding balance of the loose abrasive grain, which is likely affected by the porous wood structure.     

Atomic force microscopy of histological sections using a chemical etching method

Physiology and pathology have a big deal on tissue morphology, and the intrinsic spatial resolution of an atomic force microscope (AFM) is able to observe ultrastructural details. In order to investigate cellular and subcellular structures in histological sections with the AFM, we used a new simple method for sample preparation, i.e. chemical etching of semithin sections from epoxy resin-embedded specimens: such treatment appears to melt the upper layers of the embedding resin; thus, removing the superficial roughness caused by the edge of the microtome knife and bringing into high relief the biological structures hidden in the bulk. Consecutive ultrathin sections embedded in epoxy resin were observed with a transmission electron microscope (TEM) to compare the different imaging properties on the same specimen sample. In this paper we report, as an example, our AFM and TEM images of two different tissue specimens, rat pancreas and skeletal muscle fibres, showing that most of the inner details are visible with the AFM. These results suggest that chemical etching of histological sections may be a simple, fast and cost-effective method for AFM imaging with ultrastructural resolution.     

Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells

Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post‐embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze‐drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze‐drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.     

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.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the structural and immunocytochemical examination of biological specimens by electron microscopy

A simple and reliable method to make resinless sections for electron microscopy was recently developed by using polyethylene glycol (PEG) as a transient embedding media. In this paper the practical procedure of this PEG method is described in detail. Normal ultrastructure of several types of in-situ cells in resinless sections is demonstrated. The cytoplasmic matrix of all in-situ cells examined is revealed to consist of the microtrabecular lattice. A result from application of this technique to immuno-electron microscopy is also illustrated. This method is shown to have potential in overcoming the problem of intracellular penetration of macromolecular antibodies. Several artifacts caused by failures in specimen preparations are displayed. The real or artifactual nature of the microtrabecula is briefly discussed.     

A rapid method for the preparation of yeast for immunoelectron microscopy using lowicryl hm-20

We describe a fixation and embedding procedure for the yeast Saccharomyces cerevisiae using Lowicryl HM-20 which is rapid, gives excellent fixation, and avoids the low temperature handling normally associated with embedding in this resin. This procedure yields superior structural preservation when compared to the commonly used rapid embedding procedure which employs Lowicryl K4M. We demonstrate that sections prepared using our rapid procedure are suitable for use in immunogold labelling experiments.     

Estimation of section thickness and quantification of iron standards with EELS

The possibilities of using electron energy-loss spectroscopy (EELS) for quantification of elemental concentrations in ultrathin sections are examined. Dynabeads, which are polystyrene beads with a known iron content, are proposed as internal iron standards. The quantity of an element present depends on the thickness of the specimen. A prerequisite for estimation of absolute section thicknesses with EELS is the knowledge of the mean free path λ of electrons in the specimen. This factor is determined for the embedding resins Epon and Nanoplast by comparing EELS data with directly observed thicknesses in re-embedded sections. Dynabeads were found to include iron in a homogeneous distribution and to be stable in the electron beam.     

Observations on the ultrastructural preservation of the nucleus in the myxomycete Physarum polycephalum as observed in resinless sections

The ultrastructural preservation of the nucleus in both myxamoebae and macroplasmodia of the slime mold Physarum polycephalum was investigated using specimens processed with diethylene glycol distearate or Plexiglass. Following specimen embedding in either of these media, the polymers were dissolved from the semithin sections, which were then dried by the critical point process. In these resinless preparations, nuclei appeared to be pervaded by a complex network of fibrils of various diameters among which granules of different sizes were scattered. At least five different types of fibrils were detected in plasmodial as well as in amoebal nuclei. Resinless preparations obtained from diethylene glycol distearate processed specimens showed a number of easily recognized artifacts, some of which were noticeably less conspicuous when samples were embedded in Plexiglass. In both cases, no indication was obtained that extensive extraction of material had occurred during resin removal by solvents. The various artifacts that were produced following processing with these two media thus seem to have mostly developed during infiltration and hardening, rather than during dissolution of the polymers or critical point drying of the sections.     

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry,PCR, and fish analysis

Molecular analyses such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are demanded to improve diagnostic accuracy in addition to immunohistopathology of bone marrow (BM) trephine specimens. Conventional BM embedding method needs decalcification, and its procedure may impair tissue morphology and DNA quality. Here, we report an undecalcified method by which glycol methacrylate resin is polymerized at low temperature (4°C). Using this method, BM enzyme activity and antigenic determinants are well preserved, and moreover, DNA extracted from plastic embedding sections is suitable for PCR amplification and sequencing, FISH analysis can be well done because of the DNA integrity of BM sections. If working with BM trephine specimen, our protocol offers the possibility to combine superior morphology with modern molecular analysis. Microsc. Res. Tech. 73:1067–1071, 2010. © 2010 Wiley‐Liss, Inc.     

Comparison of polyethylene glycol and diethylene glycol distearate embedding methods for the preservation of fungal cytoskeletons

Hyphae of the fungus Basidiobolus magnus were embedded in extractable polyethylene glycol (PEG) or diethylene glycol distearate (DGD) to prepare embedment-free sections in order to seek components of the cytoskeleton that may be obscured in epoxy-embedded sections. All methods showed that hyphae possess an intricate filamentous matrix, which is apparently unoriented. However, the appearance of the cytoskeleton depended on the embedding medium, the solvent used during embedding, and whether cells were fixed by conventional fixation or freeze-substitution. PEG proved to be the best embedding medium for fungal cells because DGD caused cell shrinkage and produced a more lamellar than filamentous matrix. Also, the cytoskeletal filaments were clearer in freeze-substituted cells than in conventionally-fixed cells. Since we failed to find microtubules in the embedment-free sections, we re-embedded cells in Epon to discern whether microtubules or other cytoplasmic components had changed. Neither PEG nor DGD adequately preserved microtubules as compared to regular Epon-embedded sections, and other cellular structures were significantly altered. Therefore, alternative methods need to be employed to further characterize fungal cytoskeletons.     

Capsule-supporting ring: a new device for resin embedding of glass-mounted specimens

The goal of specimen preparation for transmission electron microscopy is to obtain high-quality ultra-thin sections with which we can correlate cellular structure to physiological function. In this study, we newly developed a capsule-supporting ring that can be useful for resin embedding of glass-mounted specimens. The present device allowed us to re-embed a semi-thin section on a microscope slide into a resin block not only for efficient ultra-thin sectioning but also for a correlative light and electron microscopy. Similar to epoxy resins for morphological observations, semi-thin sections of low-viscosity hydrophilic resins, such as Lowicryl series, can be re-embedded into the resin, which can be useful for cytochemical gold labelling. A further application of the present device improved flat embedding of cultured cells on glass cover slips for electron microscopy, preserving in situ sub-cellular structures close to their native state. We practically describe the use of capsule-supporting ring and demonstrate representative micrographs as results.     

Sectioning of Epon blocks in the 20 m to 60 micromicron range for histological studies.

Thick sections of tissue, (20 micron--60 micron), are useful in studying the relationship between individual large cells and cell layers in organized neural structures. The ability of the Nomarski Differential Interference-Contrast Microscope to bring a single thin layer into sharp focus makes the examination of such sections feasible. Although celloidin is the classical embedding medium for large, thick sections of neural tissue, the time necessary for this preparation is most inconvenient. Epon is an excellent embedding medium; however, it is extremely hard and brittle. By heating the Epon block face, thick sections can be cut. To avoid the cumbersome, often detrimental use of heat, a modification of this technique was found. Epon blocks, trimmed to a 1 millimeter square face, may be sectioned at room temperature on the sliding microtome at 20 micron to 60 micron with ease. The simple method of preparing such sections is described.     

Visualization of vesicles in semi-thin sections by the heat removal of epoxy resins.

A partial removal of the embedding medium from sections of tissue with hollow structures results in the formation of phase object. The removal of epoxy resin by the heat of a gas-burner flame is a simple method which does not lead to shifts and losses of sections from glass slides.     

Optimization of specimen preparation of thin cell section for AFM observation

High resolution imaging of intracellular structures of ultrathin cell section samples is critical to the performance of precise manipulation by atomic force microscopy (AFM). Here, we test the effect of multiple factors during section sample preparation on the quality of the AFM image. These factors include the embedding materials, the annealing process of the specimen block, section thickness, and section side. We found that neither the embedding materials nor the temperature and speed of the annealing process has any effect on AFM image resolution. However, the section thickness and section side significantly affect the surface topography and AFM image resolution. By systematically testing the image quality of both sides of cell sections over a wide range of thickness (40-1000 nm), we found that the best resolution was obtained with upper-side sections approximately 50-100 nm thick. With these samples, we could observe precise structure details of the cell, including its membrane, nucleoli, and other organelles. Similar results were obtained for other cell types, including Tca8113, C6, and ECV-304. In brief, by optimizing the condition of ultrathin cell section preparation, we were able to obtain high resolution intracellular AFM images, which provide an essential basis for further AFM manipulation.     

A simple method for exposing plastic embedded structures for scanning electron microscopy

Surfaces of structures which may be identified in sections of plastic embedded tissue can be exposed for examination using scanning electron microscopy by removal of the embedding medium from the sectioned block with sodium methoxide. Correlation of the information provided by sections and scanning electron microscopy is therefore possible. This technique has been used in examining the developing peristome teeth of the moss Funaria hygrometrica.     

Neuronal imaging with colloidal gold

Colloidal gold is easily prepared, and readily adsorbs to a number of immunoreagents and other proteins for a wide variety of uses for neuronal visualization. Gold probes serve a role as immunolabels for both light and electron microscopy. As an ultrastructural immunocytochemical marker for detection of proteins, peptides or amino acids, gold can be used for immunostaining thick or thin sections prior to embedding, or for immunostaining ultrathin sections after embedding tissue in conventional or unusual embedding matrices. By virtue of its particulate nature, gold as an immunolabel facilitates a semi-quantitative analysis of relative antigen densities on ultrathin sections. Various combinations of different size gold particles or dual immunolabelling with enzymatic immunolabels together with colloidal gold or silver-intensified gold serve well for ultrastructural immunocytochemical localization of two antigens in the same tissue section. Colloidal gold can be detected with light microscopy, transmission and scanning electron microscopy, and with confocal laser microscopy. Silver intensification allows detection of gold at both the light and electron microscope level, and increases the sensitivity of immunogold procedures. Colloidal gold is useful as a tracer for physiological studies of transport and internalization in neurons in vivo and in vitro; computer-assisted video imaging techniques allow detection and tracking of single gold particles in living cells.     

Image formation from alloys in the field-ion microscope

Previous studies of solid-solution alloys with the field-ion microscope are reviewed, and it is pointed out how their image contrast must be determined by the different response of the atomic species towards field ionization and field evaporation. The former effect, termed selective ionization, describes the relative brightness of the image points arising from the different atomic species. It is pointed out that this process is secondary to considerations of selective evaporation since the latter determines whether both species can occupy potential imaging sites in any given region of the image. Selective evaporation describes the relative fields required to evaporate the two species of atom from the specimen surface. In general the solute species will either be preferentially evaporated from the surface defined by the solvent species, or will be preferentially retained onto it. Consideration of the mechanism of selective evaporation shows that its degree may be expressed by a simple parameter calculated from known thermodynamic and atomic-bonding data. In the later sections the model is applied to reported field-ion data from solid-solution alloys, excellent correlation being obtained.