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).     

The Ralph knife in practice

Long edge glass knives, ‘Ralph knives’, were produced in an LKB Histo KnifeMaker. The edge angles were measured by light microscopy; depending on the breaking conditions the angles varied between 12° and 58°, as measured close to the edge in the mid-portion of the knives. Hackle marks were more common in the left portion of the edge than in the middle and right portions. Some of the knives were used for cutting sections from urinary bladder tumours embedded in paraffin or in glycol methacrylate. Following microtomy the sections were allowed to stretch on a water surface; this procedure resulted in an increased width of the plastic sections, whereas the paraffin sections were not affected. The compression of the whole sections averaged 15% in the paraffin sections and 11% in the plastic sections; in both cases the compression factors were positively correlated to the angle of the knife edge. Smaller compression factors were found for the cell nuclei in the embedded tissue.     

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.     

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.     

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.     

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.     

Cutting teeth in the SEM

An SEM was used to observe and record dental tissues as they were being cut. Sound human deciduous and permanent teeth were stored in 70% ethanol until required, then soaked in water, superficially dried and screwed to the SEM specimen stage through small drill holes made when they were wet. Many specimens were frozen and studied at cryogenic temperatures so that they would not become dehydrated. Edges used to cut the teeth included steel and tungsten carbide fashioned to resemble clinical cutting instruments, and diamond ultramicrotome knives and burs. The cutting tools were held either in a micromanipulator or a rigid tool post clamped to the specimen stage. The finest control was obtained by moving the specimen with the usual stage controls. SEM was conducted at 3 or 5 kV using TV speed scanning on the uncoated samples. All experiments were video-taped. 3-D control was difficult with a mono image and a real-time stereo system was therefore developed. Continuous, flowing sections of enamel could be obtained using diamond knives to cut the prism-free, surface zone tangentially. Thin sections of dentine, cement and bone curled up as they were cut, thus demonstrating permanent deformation. Subsurface enamel always fractured as it was cut, either locally as the tissue passed over the knife edge or tore out beneath the plane of the knife or by larger fragments cleaving off at larger distances ahead of the knife. Appearances were characteristic of prism orientation with respect to cutting direction. No anisotropy of cutting behaviour was found with dentine or bone: These tissues only fractured when thicker sections were taken. The SEM methods employed here can be usefully applied in the study of other materials.     

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.     

新型果蔬切丁机的设计

国内现有切丁机存在着结构复杂、生产效率低、切出10mm以上果蔬大丁形状不规整的问题,无法满足国内果蔬加工业的发展需求。提出采用离心切削法进行果蔬切片、圆盘刀切丝、横切刀切丁的三维切丁技术的新型果蔬切丁机方案。通过对果蔬的切丁成型分析,得到切10mm以上果蔬大丁形状不规整的原因。仿真分析得到切丁机推进器和横切刀之间的速比以及两者之间的中心位置布置的合理组合。对切丁机主要结构部件进行了设计,研制果蔬切丁机样机。性能试验表明:切出的(15×15×15)mm和(20×20×20)mm马铃薯立方块形状规整,其相对百分数均小于15%,达到了设计要求。     

A synthetic corundum knife for ultrathin sectioning*

A synthetic corundum (Al₂O₃, synthetic ruby, synthetic sapphire) knife has been constructed which is suitable for the preparation of thin sections (less than 90 nm) of biological specimens for electron microscopy. The method involved cleavage or fracture of the flame-fusion crystalline product, followed by mounting in a standard knife holder for ultramicrotomy. The cutting edge appeared flawless under 400 × and produced sections in which subcellular organelles were clearly resolved. Scanning electron microscopy of the knife revealed conchoidal fractures typical for corundum, terminating in apparent cleavage of the crystal along the knife edge. X-ray microanalysis of the knife edge revealed essentially 100% aluminium, indicating that the aluminium oxide crystal was of a high purity. The superior toughness and low cost of synthetic corundum suggest that it may be a useful alternative to diamond or glass knives.     

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.     

A simple microtome capable of cutting sections of plastic-embedded material down to 1 μm in thickness

Details of the design of an easily constructed microtome are presented. The instrument uses glass knives to cut sections down to about 1 μm in thickness, and is suitable for the examination of plastic-embedded material.     

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

The geometry of glass knife edges for ultramicrotomy was studied with nanoscale resolution using scanning force microscopy (SFM) in the contact mode. The local shape of the cutting edge was estimated from single line profiles of the SFM topographic images by taking into account the exact radius of the ultrasharp silicon tip. The tip radius was estimated from secondary electron micrographs recorded at low voltage by field emission scanning electron microscopy (FESEM). The radius of the investigated cutting edges was found to be in range 5–20 nm. The results obtained illustrate that the combination of SFM and high resolution FESEM provides a unique means to determine precisely the radius of glass knives.     

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.     

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.     

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.     

Electron-microscope preparation techniques applied to the light microscopy of the cambium and its derivatives in Pinus radiataD. Don.

Epon 812 was used successfully as an embedding medium for the preparation of the secondary meristem and its derivatives in Pinus radiata for light microscopy. Specimens were fixed in glutaraldehyde followed by osmium tetroxide solution, using a schedule developed for the electron microscopy of these tissues. Sections from 2 to 5 μm thick were cut using an LKB Ultratome III and glass knives. Sections were evaluated without prior removal of the embedding medium using bright-field, phase-contrast and interference-contrast microscopy. Results for cambium were generally superior to those obtained by using the conventional fixatives for light microscopy and paraffin embedding. The advantages and disadvantages of this technique are discussed and it is concluded that the use of Epon for embedding this type of material is easier and gives better results than older established methods. It is suggested that methods of electron microscopy be considered when the preparation of a material for light microscopy has proved difficult or impossible by other means, or when particularly thin sections are required.     

Cutting wood specimens for observation in the scanning electron microscope

This report deals with the cutting of wood specimens for observation in the scanning electron microscope. Several cutting devices and types of knives are described and critically evaluated.     

金属切削中如何选择切削液

概述了切削液的种类和性能,介绍了目前切削液的使用情况,重点探讨了在不同的材料、不同的切削方法、各种常见刀具及良好的经济效益下选择切削液的基本原则.     

Megavolt and cryo electron microscopy of diamond knife edges

As part of a comprehensive research program, observations of the primary cutting edge and facets of representative types of diamond knives have been made by low-temperature electron microscopy at 100 keV, and by HVEM at 1 MeV. Standard knives prepared from selected Venezuelan alluvial diamonds, which were subjected to cryogenic microirradiation techniques, show improved ultrasharp cutting edges (radii of 2–4 nm) and characteristic layered period structures of the facets. At 1 MeV the higher penetration power reveals novel features of the wedge-shaped facet and edge. Dark field images show equal-thickness fringes and previously undetected diffraction contrast patterns of imperfections in the crystalline lattice introduced during the sharpening process. These correlated studies are essential for rational control and improvement of diamond edge quality which is a critical element for ultramicrotomy and precision machining of optical surfaces. They also illustrate the potential utilization of other unique properties of diamond by related synergistic techniques.