Correlation of some mechanical properties of embedding resins with their behaviour in microtomy

Abstract

The technology of ultramicrotomy is now well established, but the properties of the resin that determine the different forces needed to generate a section have been neglected, although this process could introduce artefacts in the thin sections. We have investigated the principal resin dependent factors involved in the sectioning process and determined the related mechanical properties. Tensile experiments have given the best correlation with the sectioning quality of the resin: the elastic (Young's) modulus value (depending on polymer structure or hardening mode), the presence of a short plastic flow for a controlled fracture and enough flexibility to minimize shearing, and internal cracks, appear to be the main characteristic parameters. The ultrathin section seems to be generated by a process close to cleavage, favoured by the relative hardness of the embedding media, while machining and "true" sectioning requires softer resins.Consequently, the rupture follows the path of least resistance in the specimen-resin composite, providing sections with a surface relief. Embedded biological material copolymerizes with polycondensed matrix (epoxy resins), and, by reducing the heterogeneity, gives smoother sections. Embedments hardened by radical polymerization provide a rougher relief, since almost no copolymerization occurs, offering to the microtome a heterogeneous block with two constituents of very different mechanical properties. The surface relief seems to be a n important factor in labelling, staining, and imaging, and more attention has to be Daid for some imDrovements of the quality of the information Plastic flow, Section surface relief provided by electron microscopy.

Learning about truth and biases through experience: Section surface corrugation, protein denaturation, and staining

“…With this new resin, the effects of surface relief on imaging were strongly enhanced (Kellenberger et al, 1986) and convincingly revealed the origin of the blurring. We could demonstrate a strong deformation of the resin surface in the near neighborhood of biological material, which was in full agreement with the physics of cleavage mentioned above and discussed elsewhere (Acetarin et al, 1987). Through cleavage, all matter of the section surface is submitted to strong stress that induces elongation through the so-called plastic flow, before rupture, and thus separation occurs (Fig.…”

“…Protein fibers (e.g., hair) will have much more plastic flow than embedding resins and, therefore, emerge on both corresponding surfaces of the consecutive sections. The physics of cleavage of biological embeddings has been explained and further developed by Acetarin et al (1987). It predicts a corrugated surface, summarized in Figure 2, and we now have to demonstrate the existence and to show the consequences of this on immunolabeling and resolution.…”

Use of Thin Sectioning (Nanoskiving) to Fabricate Nanostructures for Electronic and Optical Applications

“…Figure 1d is a schematic drawing of the process of sectioning, which involves a complicated interplay of events: compression of the sample during the initiation of cutting, and of the slab thereafter; tension perpendicular to the plane of sectioning; generation of new surfaces; bending, as the slab reorients from vertical to horizontal; shearing stress (greatest in materials with low flexibility); friction of the slabs on the knife; and generation of heat. [55] The nascent epoxy slabs slide onto the surface of a water bath in the form of individual slabs or ribbons of connected slabs. [56] There are two general mechanisms of the process of sectioning proposed in the literature of ultramicrotomy: i) true sectioning, in which the edge of the knife maintains contact with both the bottom surface of the nascent slab and the facet of the block, and ii) crack initiation and propagation.…”

“…The embedding medium should have two properties, i) a relatively high value of elastic modulus (~3 GPa; materials that are too compliant deflect from the knife edge, rather than cleave), and ii) a high yield stress, only after which the material undergoes plastic deformation (~70 MPa; otherwise the slab will deform upon sectioning). [55] Crosslinked epoxy resins fill most of these criteria at ambient temperatures, although it is possible to section softer materials at cryogenic temperatures. Our laboratory has achieved excellent results with UVO-114 (Epotek), which is UV-curable, [59] and good results with Araldite 502 [60] and Epo-Fix (Electron Microscopy Sciences), [61] which are thermally curable.…”

Image contrast in sections of epoxy resin-embedded biological material: Maintenance of a proper anhydride-epoxy ratio during tissue impregnation

“…Epoxy resins react with tissue elements through covalent links with the embedded tissue (Acetarin et al, 1987;Kellenberger et al, 1986;Luft, 1973). This is probably the primary reason that epoxy resins are such good glues, that they inhibit reactive sites of tissue elements, and that they give such good tissue preservation.…”