A model for cryosectioning based on the morphology of vitrified ultrathin sections

Abstract

SUMMARY Electron microscopy of vitrified ultrathin sections allows cell ultrastructure to be studied in the hydrated state. Sectioning of the frozen material is, however, a limiting step, since the cutting forces cause severe mechanical deformation. In order to address this problem, we have investigated the surface of cryosections. It is shown that cryosections have two fundamentally different surfaces. One surface is rough, deformed by cutting‐induced deformation lines which are orientated perpendicular to the cutting direction. The other surface, in comparison, is not affected by those deformation lines. Except for knife marks it is smooth. In order to explain the observations, the following model is proposed. The rough relief corresponds to the former block face. Its roughness originates from material that is squeezed out of the section plane when the section is compressed in the cutting direction and bent away from the specimen block. The smooth section surface is the surface in contact with the knife during the sectioning. This contact keeps the surface smooth while imprinting the knife marks.

Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy

“…There are accumulations of crevasses on the large top KM and some top KM seem to be imprinted in the crevasses (white ellipses in A and B). This fact is in line with the observation of Richter et al (1991) that crevasses are on the top-side of the section.…”

“…There are numerous articles dealing with the cryosectioning process or directly relevant to it Dubochet and McDowall, 1984;Dubochet et al, 1988;Hsieh et al, 2002;Jésior, 1986;Lickfeld, 1985;McDowall et al, 1983;Richter et al, 1991;Richter, 1994a,b,c;Sartori et al, 1993;Sartori Blanc et al, 1998;Sitte, 1996;Studer et al, 2001;Zhang et al, 2004;Zierold, 1984Zierold, , 1987) but many aspects remains uncertain or not understood. It is the aim of the present study to analyse in more detail the cutting artefacts and the cutting process and determine the optimal conditions for producing high quality vitreous sections.…”

Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections

“…In a previous report on rat liver , we described the Wrst successful application of electron tomography to frozen-hydrated mammalian tissue. Artifacts associated with cryo-ultramicrotomy include knife marks, crevasses, chatter, and compression Chang et al, 1983;Frederik et al, 1982Frederik et al, , 1984Michel et al, 1991Michel et al, , 1992Richter et al, 1991;Richter, 1994;Zierold, 1994). Our tomographic analysis revealed that the Wrst two types of defects were predominantly conWned to the section surface, and that there was considerable useful ultrastructural information in the interior of the sections .…”

Focus on Membrane Differentiation and Membrane Domains in the Prokaryotic Cell

“…Al-though we have stated before that tomography is limited to 1-μm-thick objects, this can be circumvented using cryo-sectioning (after high-pressure freezing). This technique can provide thin sections of 70 nm thickness of any kind of a large bacterium or without chemical fixation [Richter et al, 1991]. An additional EM option is elemental analysis: the normally 'useless' inelastically scattered electrons in a standard electron microscope can be correlated to an element by energy-filtered imaging.…”