Cryo-Planing Biological Specimens
membranes no longer osmotically active so that specimens can be washed in pure water, reducing the potential appear- ance of salt deposits on the specimen. Te OsO4
fixation can
also provide electrical conductivity. Osmium tetroxide fixa- tion when combined with mordents such as thiocarbohydra- zide and tannic acid can even provide sufficient conductivity to enable high-resolution SEM without metal coatings [9]. Tus, it may be possible to extend this method to minimally coated or even uncoated specimens. Tis cryo-planing methodology using mPrep capsules and
a cryo-ultramicrotome could be modified by rapid-freezing in cryogens other than LN2 slush, such as chilled ethane or propane [4,9]. Te present methodology could also be adapted for frozen-hydrated SEM imaging, wherein cryo-planed spec- imens are transferred to a cryo-coater and thence to a cryo- SEM [1,2]. While the present specimens were about 2–3 mm thick
and 5 mm long, the mPrep/s capsules readily accommodate specimens as thin as 100 μm. Smaller and thinner specimens can improve the freezing rate and thus potentially improve image quality and/or volume of the vitreous frozen region. Te capsules can also accommodate specimens that are even a few millimeters larger than those used here, but of course there are limits on the rate of freezing with larger specimens, which can have deleterious effects on the quality of the preparation of hydrated biological specimens. However, this would not be an issue with non-hydrated specimens where one wants to prepare cross sections, such as polymers, composites, pharmaceuticals, and other materials.
Conclusions Te methodology demonstrated here provides ease, qual-
ity results, and experimental flexibility for the preparation of cryo-planed plant and animal tissue specimens. Because the process is accomplished with a conventional cryo-ultra- microtome and readably available mPrep/s capsules, this method can be easily performed in many labs. Tis could enable cryo-planing to be done more easily and more rou- tinely in the examination of biological and many non-biolog- ical specimens.
References [1] J Njisse and AC van Aelst, Scanning 21 (1999) 372–78. [2] P Walther, Microsc Microanal 9(4) (2003) 279–85. [3] IY-T Chang and D Joester, Microsc Microanal 21(S3) (2015) 377–78.
[4] JT Van Dongen et al., Annals of Botany 91 (2003) 729–27. [5] Y Utsumi and Y Sano, “Cryoplaning Technique for Visu- alizing the Distribution of Water in Woody Tissues by Cryoscanning Electron Microscopy” in Electron Micros- copy: Methods and Protocols: Methods in Molecular Biol- ogy, Second Edition, Vol. 369, ed. J Kuo, Humana Press, Totowa, NJ, 2007.
[6] WC Plumley, Microsc Microanal 20(S3) (2014) 1442–43. [7] SL Goodman et al., Microscopy Today 23(5) (2015) 30–37. [8] MJ Karnovsky, J Cell Biol 27 (1965) 137–38A. [9]T Murakami et al., Scan Electron Microsc 1983(1) (1983) 235–46.
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