Measuring the resolution of uncompressed plastic sections cut using an oscillating knife ultramicrotome.
Thin sections of biological tissue embedded in plastic and cut with an ultramicrotome do not generally display useful details smaller than approximately 50 A in the electron microscope. However, there is evidence that before sectioning the embedded tissue can be substantially better preserved, which suggests that cutting is when major damage and loss of resolution occurs. We show here a striking example of such damage in embedded insect flight muscle fibres. X-ray diffraction of the embedded muscle gave patterns extending to 13A, whereas sections cut from the same block showed only approximately 50 A resolution. A possible source of this damage is the substantial compression that was imposed on sections during cutting. An oscillating knife ultramicrotome eliminates the compression and it seemed possible that sections cut with such a knife would show substantially improved preservation. We used the oscillating knife to cut sections from the embedded muscle and from embedded catalase crystals. Preservation with and without oscillation was assessed in Fourier transforms of micrographs. Sections cut with the knife oscillating did not show improved preservation over those cut without. Thus compression during cutting does not appear to be the major source of damage in plastic sections, and leaves unexplained the 50 A versus 13A discrepancy between block and section preservation. The results nevertheless suggest that improvements in ultramicrotomy will be important for bringing thin-sectioning and tomography of plastic-embedded cells and tissues to the point where macromolecule shapes can be resolved.
Sader
Kasim
K
Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Reedy
Michael
M
Popp
David
D
Lucaveche
Carmen
C
Trinick
John
J
D000818:Animals; D002374:Catalase; D006801:Humans; D008867:Microtomy / Q000295:instrumentation* / Q000379:methods; D009132:Muscles; D010969:Plastics / Q000737:chemistry*; D016610:Tissue Embedding / Q000379:methods* / Q000592:standards; D014021:Tissue Preservation; D014961:X-Ray Diffraction
D010969:Plastics; D002374:Catalase