An oscillating cryo‐knife reduces cutting‐induced deformation of vitreous ultrathin sections

Abstract

SummaryA new oscillating cryo-knife for producing uncompressed vitreous sections is introduced. The knife is a modified cryo diamond knife that is driven by a piezo translator. Optimal setting for the oscillation was found to be in the inaudible frequency range of 20 -25 kHz. Yeast cells and polystyrene spheres were used as model systems to describe compression in the vitreous sections. We found that compression could be reduced by a factor of about 2 when the knife was oscillating. When the oscillator was turned off, sections were compressed by 40 -45%. However, only 15-25% compression was obtained when the knife was oscillating. In some cases completely uncompressed sections of yeast cells were produced. It was also found that the amount of compression depends on the specimen itself and on its embedding medium. With the results shown here, we demonstrate that the oscillating knife can produce high-quality vitreous sections with minimum cutting artefacts.

Architecture of Thylakoid Membrane Networks

“…Future holds promise for turning CEMOVIS into a more reproducible and, hopefully, artifact-free process. The sectioning process may be improved by refining conventional sectioning techniques (Han et al, 2008), as well as by utilizing new instruments and techniques, such as oscillating diamond knives (Al-Amoudi et al, 2003) or focused ion beam milling . It is also hoped that the inherently poor contrast of frozen sections will be enhanced by the utilization of phase plates (Nagayama and Danev, 2008), which would eliminate the need to work at large underfocus values in the transmission electron microscope in order to obtain reasonable contrast.…”

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

“…It involves the shortening and thickening of sections and leads to a loss of resolution. To minimise this problem, it is recommended to decrease the knife angle [40] and keep the diamond knife scrupulously clean [41]. The application of an oscillating cryo-knife is an alterna- www.fhc.viamedica.pl tive way to reduce compression [40].…”

Measuring the resolution of uncompressed plastic sections cut using an oscillating knife ultramicrotome

“…Sections were cut with a Leica UCT Ultracut ultramicrotome fitted with an oscillating diamond knife (Al- Amoudi et al, 2003;Studer and Gnaegi, 2000). A 50 nm per cutting cycle feed and a cutting speed of 0.4 mm/s were used.…”

“…Thus, if compression could be eliminated, preservation might be greatly improved. An oscillating ultramicrotome knife has been demonstrated to eliminate the compression in plastic sections (Al-Amoudi et al, 2003;Studer and Gnaegi, 2000), but the effect of this on resolution has not been measured. Here we report data from sections cut with and without the knife oscillating, from the same blocks of embedded insect flight muscle that were shown to give X-ray transforms extending to 13 Å .…”