Some observations on knife properties and sectioning mechanics during ultramicrotomy of plastic embedding media

The edge and the trough surface of ultramicrotome glass knives were studied by interference light microscopy and electron microscopy. Particular attention was paid to the region which is commonly used for microtomy; here the bevel angle was calculated to 50–55°. The edge was serrated, even in the region used for sectioning, where the indentations measured up to 20 nm in depth. The imprints of edge serrations left on the sectioned material (Vestopal) were studied by electron microscopy. Different embedding materials displayed different degrees of compression when sectioned in the ultramicrotome: with Epon the compression was less than 10%, whereas methacrylate was compressed by more than 30%. Different trough fluids were tested: aqueous solutions of ethanol, acetone and dioxane gave less compression than pure water. However, the compression could also be reduced by exposing compressed Vestopal sections to these solutions. The direction and magnitude of the cutting force was measured with transducers during the sectioning of Vestopal blocks. Thus ～0·3 μm wide sections cut at a feeding rate of 80 nm and a speed of 2 mm/sec required a cutting force of about 8 mN. The direction of the forces was 8–15° from the plane of cutting.     

Characterization of the cutting edge of glass and diamond knives for ultramicrotomy by scanning force microscopy using cantilevers with a defined tip geometry. Part II

The cutting edge of glass as well as diamond knives was studied at high resolution using a scanning force microscope (SFM). The local shape of the cutting edge was estimated from single line profiles of the SFM topographs taking into account the exact shape of the probing tip estimated by a high‐resolution field emission scanning electron microscope (FESEM). The glass knives were prepared by ‘balanced breaking’. The radius of the investigated cutting edges was found to be 3.2–4.4 nm and 4.3–6.0 nm for the 35° and 45° diamond knife, respectively, and 3.4–4.3 nm for the glass knives. Besides the opening angle and the cutting edge radius, the friction of a knife during sectioning represents a significant factor influencing the quality of sections. Thus, the roughness of both the diamond clearance angle side and the back side was characterized as well. Corresponding RMS values of the roughness were found to be smaller on the back side (≈ 0.14 nm) than on the clearance angle side (≈ 0.26 nm).     

Modified paraffin wax for improvement of histological analysis efficiency

Paraffin wax is usually used as an embedding medium for histological analysis of natural tissue. However, it is not easy to obtain enough numbers of satisfactory sectioned slices because of the difference in mechanical properties between the paraffin and embedded tissue. We describe a modified paraffin wax that can improve the histological analysis efficiency of natural tissue, composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 5, and 10 wt %). Softening temperature of the paraffin/EVA media was similar to that of paraffin (50–60°C). The paraffin/EVA media dissolved completely in xylene after 30 min at 50°C. Physical properties such as the amount of load under the same compressive displacement, elastic recovery, and crystal intensity increased with increased EVA content. EVA medium (5 wt %) was regarded as an optimal composition, based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices, amount of load under the same compressive displacement, and elastic recovery test. Based on the staining test of sectioned slices embedded in a 5 wt % EVA medium by hematoxylin and eosin (H&E), Masson trichrome (MT), and other staining tests, it was concluded that the modified paraffin wax can improve the histological analysis efficiency with various natural tissues. Microsc. Res. Tech. 73:761–765, 2010. © 2010 Wiley‐Liss, Inc.     

Surfactants as resin modifiers and their effect on sectioning

Ease of cutting thin sections with glass knives is markedly improved if the embedding resin contains a surfactant such as lecithin. With lecithin, it is possible to cut 50–100 thin sections from the same place on the knife edge even after facing off the block with 1–2-μm-thick sections. Image quality is similar to that of the unmodified resin if the resin formula is optimized. If not, some chatter or a “mottled” appearance of the tissue image may be present. Lecithin does not significantly affect sectioning with a diamond knife or the appearance of the section in the microscope. The increased ease of sectioning with glass presumably will be translated to diamond knives in the form of an increased useful life of the cutting edge.     

Vitreous cryo-sectioning of cells facilitated by a micromanipulator

Sectioning vitrified cells and tissues for cryo‐electron microscopy is more challenging than room‐temperature sectioning of plastic‐embedded samples. As the sample must be kept very cold (相似文献   

Diamonds are a cryosectioner's best friend

High-pressure frozen Golden Delicious apple leaves were cryosectioned at low temperature with diamond knives. Good cryosections were obtained by optimizing the cutting parameters, i.e. sectioning temperature, mechanical stability of the sample, and sectioning velocity. Cutting artefacts were minimized by reducing the electrostatic interactions between the knife surface and the cryosection. This was accomplished by sectioning the sample in the presence of an ionization electrode. The ionization device, with a primary voltage of 7–8 kV, produces positively and negatively charged nitrogen ions which neutralize the surface charges of the knife and the section. This minimizes the friction on the knife surface and results in ultrathin sections without crevasses or knife marks. Compression of the sections could be minimized, but not avoided, by reducing the knife angle to 30°. Improved contrast of the frozen-hydrated sections was obtained with the Zeiss EM 902 energy-filter microscope operated in the zero-loss mode.     

Thickness variations within individual paraffin and glycol methacrylate sections

The object of this study was to investigate whether there are intra-section thickness variations in individual paraffin and glycol methacrylate (GMA) sections. Using steel or glass knives sections were cut from liver and urinary bladder. Section thickness variations were measured with an interference microscope and amounted to 1–3 μm within individual paraffin sections and 0.3 μm within GMA sections. The results were confirmed by observations on sections which had been re-embedded and re-sectioned. Some of the variations within the paraffin sections were associated with the cell nuclei. It is concluded that GMA sections are much smoother than paraffin sections and thus more suitable for quantitative histological studies.     

One micrometre paraffin sections: an aid to interpretation of immunoperoxidase staining of immunoglobulins

A technique is described for obtaining 1 μm sections of tissue embedded in modified paraffin wax using long-edged glass knives. Serial sections can be cut and stained by the immunoperoxidase method to demonstrate multiple antigens in a single cell or to confirm that the immunoglobulin within a cell is monotypic. The improved cytological detail seen in thin paraffin sections permits the more precise localization of intracellular staining.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

An anti-roll plate system for Ralph knives

At thicknesses above 4 μm, an anti-roll plate is necessary when cutting frozen sections for light microscopy with long-edged glass (Ralph) knives. The structure of the plate and a system which allows the use of such plates without dismantling or interfering with present systems for steel knives is described.     

Steedman's polyester wax embedment and de-embedment for combined light and scanning electron microscopy

Steedman's polyester wax mixture is a good, general-purpose histological embedding medium that is suitable and convenient to use when it is desirable to combine light microscopy with scanning electron microscopy (SEM). A range of properties recommend this wax: it has a low melting temperature (37°C), is readily soluble in most dehydrating agents, results in negligible tissue shrinkage, preserves tissue antigenicity, and may even be used as a solvent for fixative agents. We prepare and embed tissues in polyester for light microscopy much as they would be for paraffin wax. For SEM, the block surface is micro- or ultraplaned, utilizing, respectively, a standard rotary microtome with razor blade knives or an ultramicrotome with glass knives. The block is de-waxed in absolute alcohol and then taken to critical point drying. Similarly, sections mounted on coverslips or glass slides may be brought to the SEM after removing the wax. This enables one to bring to the SEM relatively large block faces or sections with good control over orientation. We find the results to be superior to similar procedures employing paraffin. We believe it to be more versatile and equivalent or superior to a variety of other techniques designed to gain access to the interior of tissues with SEM.     

Surfaces of cryosections: is cryosectioning ‘cutting’ or ‘fracturing’?

In order to determine if cryosectioning involves ‘fracturing’ or ‘cutting’ we examined the surfaces obtained in cryosectioning by a metal-replicating procedure commonly used in freeze-fracture microscopy. Platinum-carbon replicas were made of the surfaces of both the sections and the complementary surfaces of the sample stubs from which the sections were cut. When samples of frozen red cells were sectioned at ?120°C with large knife advancements (1 μm), the chips produced did not resemble sections. Membrane fracture faces, produced by splitting of the lipid bilayer, were found in electron micrographs of replicas of the sample stubs. This demonstrates that a cryomicrotome can be used to produce large intact replicas. When dull knives were used with small knife advancements, both smooth and fractured regions were found. The sections produced with dull knives had a snowflake appearance in the light microscope. When sharp knives were used with small advancements (0·1 μm), replicas of the surfaces were free of fracture faces and the sections had a cellophane-like appearance in the light microscope. Therefore, in cryosectioning a different process other than ‘fracturing’ is responsible. This ‘cutting’ process may be micromelting of a superficial layer by the mechanism of melting-point depression from the pressure exerted by the sharp edge of the knife.     

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.     

一种非接触式的刀口角测量系统

为了测量五金刀剪的刀口角度,在流体接触式电容倾角传感器的基础上,设计并制造了一种基于激光的非接触刀口角测量系统。该系统结构简单,可实现刀口角的非接触测量。通过对两种刀的刀口角测量表明,该系统测量刀口角的误差小(约为0.3°),精度高(0.1°),特别适用于刀剪行业的刀口角度检测。     

A technique for obtaining ribbons of epoxy and other plastic sections for light microscope histology

A method is described by which ribbons of thick, large area sections of material embedded in epoxy resins prepared from standard recipes for electron microscopy, can be cut using conventional microtomes. The epoxy blocks are double embedded in an epoxy/polyethylene glycol mixture and ribbons are cut dry with fluorocarbon coated long-edged (‘Ralph’) glass knives. The method can also be applied to other plastic embedding media such as glycol methacrylate.     

A simple modification to the LKB 7800 series Knifemaker and a balanced-break method to prepare glass knives for cryosectioning

A novel method of using the LKB 7800 series Knifemaker to produce glass knives using a balanced break is described. The method produces knives of sufficient quality to section aldehyde-fixed, sucrose-cryoprotected, frozen biological material in a cooled cryochamber of an ultramicrotome. The modifications to the Knifemaker are minimal and, if required, the machine can be returned easily to its normal state after use.     

Minimal compression of ultrathin sections with use of an oscillating diamond knife

With the aim to minimize compression artefacts in ultrathin sections, coincident with the stroke direction, we have invented an oscillating diamond knife. Results and theoretical considerations explaining its function are discussed. During conventional ultrathin sectioning the resultant compression is in the order of 20–35% of section height. This holds true for sections of samples embedded into Lowicryl HM20 and of the polymer polystyrene, cut with a 45° diamond knife and floated on water. The oscillating knife reduces this compression almost completely. It consists of a diamond knife on which a low voltage piezoelectric translator (piezo) is mounted, which oscillates when the piezo is driven by an alternating voltage source. No additional cutting artefacts were observed in the micrographs when they were compared with sections produced without oscillating the knife.     

Tungsten coating--a method of improving glass microtome knives for cutting ultrathin sections.

Glass knives for ultramicrotomy were considerably improved by coating the cutting edge with a film of evaporated tungsten metal. Knives treated by this method gave up to tem times as many acceptable sections as uncoated glass knives. They could also cut thinner sections and harder tissues than ordinary glass knives, and eliminated some of the cutting artefacts produced by them. No explanation for this improvement was found although several possibilities were examined.     

Spatial orientation of arterial sections determined from aligned vascular smooth muscle

The alignment of the arterial axis can be used as the required reference for three-dimensional (3-D) measurements of structures within the artery wall. Our hypothesis is that this alignment reference may be derived mathematically if one uses medial smooth muscle, a tissue component we have found to have a circumferential organization in human cerebral and coronary arteries. We tested this hypothesis for angles of sectioning up to 60°, using arteries fixed at normal distending pressure. These segments of artery were sectioned at precisely measured angles, using a specially designed mitre box, and the sections were stained to enhance birefringence of the smooth muscle. Arteries from eleven autopsies were obtained from the heart and brain, and measurements of 3-D orientation on medial smooth muscle were made with a polarizing light microscope equipped with a four-axis Universal stage. By comparing the cutting angle, derived from our mathematically obtained values, to the predetermined cutting angle, we deduce that we can determine the obliqueness of cut relative to the arterial axis to within 4.6°. The application that is important to us, and possibly to other laboratories, is that the necessary direction reference for an artery is completely contained within a single tissue section.     

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.