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

Abstract

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 loo!,, whereas methacrylate was compressed by more than 30o/b. 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 pm 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.

An anti‐roll plate system for Ralph knives

“…In attempts to improve the cutting process and knowledge of it, compression (Dempster, 1942a(Dempster, , by 1943, section thickness (Hallen, 1956;Williams & Meek, 1966;Wachtel et al, 1966), surface topography of sections and blocks (Helander, 1969), cutting forces (Helander, 1974;Saubermann et al, 1977) and knife angles (Hallen, 1956;Marengo, 1967;Ward, 1977) have all been investigated. Their findings have not altered common practice, however, and the cutting of sections remains a skilled process which is not codifiable (Gabe, 1976).…”

The Ralph knife in practice

“…Under ideal conditions the comparable figure in Halltn's investigation amounted to lO-lS;/,. The degree of compression was demonstrated to depend on, among other things, the edge angle, the section thickness and the ambient temperature. The compression of ultrathin plastic sections for electron microscopy has been shown to vary between 7% and 33% (Helander, 1974;Peachey, 1958).…”

Monitoring cutting forces with an instrumented histological microtome

“…A number of studies have been made on the forces involved in cutting, both of metals in a lathe (Okoshi, 1930) and of specimens in a microtome (Helander, 1974;Saubermann et af., 1977). However, both attempts to measure forces during microtomy suffered from too much compliance (i.e.…”

“…However, both attempts to measure forces during microtomy suffered from too much compliance (i.e. insufficient stiffness) either in the knife mounting (Helander, 1974) or in the mechanism used to move the specimen up and down (Saubermann et al, 1977). Recently, Ericson & Lindberg (1994, 1995 have successfully measured work of cutting of ultrathin sections of plastic using a piezo element in the sample holder.…”

“…This militates against the received wisdom that both specimen and knife must be mounted as rigidly as possible, although there is no published information as to how stiff the mounting of the knife or specimen must be. Helander (1974) chose to modify the knife holder of an ultramicrotome but made no observations on the compliance of the holder. He found forces of the order of 10-50 mN during the sectioning of resin blocks, but had to use a very compliant knife mounting (maximum 0.14 p m r n~-' , about 2000 times more compliant than the one we used).…”