Concerning the nature of the cryosectioning process

Abstract

Intentional melting of frozen-thin-sections induces ultrastructural alterations. Cross sections through frozen dried and embedded cryosections revealed that even at the surface of such a section no melting artefacts are visible. One surface of the cryosection (obtained at 168 K) has a saw-teeth appearance whereas the other surface is rather smooth. The rate of freeze-drying in the cryochamber of the microtome was estimated to be in order 3.3 nm/min at 183 K and 22.5 nm/min at 193 K for sections, about 120 nm thick, of rat kidney frozen in Freon 22 without any chemical fixation or cryoprotection. Although freeze-drying will normally not interfere with the cryo-sectioning process itself, it is a process which cannot be neglected during handling of sections. The available evidence indicated that frozen-thin-sections represent the frozen tissue with respect to the ultrastructure and probably also with respect to the chemical composition.

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 .…”

“…the gold particles, are magniWed in the section when it is some distance from the plane of the markers. In an attempt to improve section Xatness and induce attachment, we routinely press sections onto grids with a glass section press (see Section 2) or a metal rod (Frederik et al, 1982;McDowall et al, 1983). While these procedures may Xatten the sections to some degree, results are not consistent.…”

Freezing, sectioning and observation artefacts of frozen hydrated sections for electron microscopy

“…Attempts have been made to understand the sectioning process as a fracture in a solid or as a deformation in a plastic material (see Saubermann, 1980;Frederik et al, 1982). The aspect of many replicated frozen sections or block surfaces which are similar to freeze-fractured preparations supports the fracture theory.…”

Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

“…Currently, the methods used for creating attachment involve mechanical forces, generated by either a stamping tool or a pressing device (Christensen, 1971;Frederik et al, 1982). Using these methods for attachment, damage in the form of large fissures or cracks can be observed , which alters the integrity of both the ribbon of vitreous cryo-sections and the individual sections.…”