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Cryo-Planing Biological Specimens for Scanning Electron Microscopy

  • William C. Plumley (a1), Michael Kostrna (a1) and Steven L. Goodman (a2)
Abstract

Cryo-planing SEM provides a powerful 3D view of internal and external tissue and cell features. However, specimen preparation is tedious because it requires custom apparatus and difficult cryogenic manipulations. We present an easy method using mPrep/s capsules that provide a “handle” to hold and orient specimens throughout cryo-preparation and imaging. Cryo-facing is done with a cryo-ultramicrotome, requiring no custom equipment while providing accurate control of the image plane. We show this with fresh Christmas cactus leaves and aldehyde-fixed kidney tissue imaged by room temperature SEM after freeze-drying, demonstrating the method operating without an expensive cryo-SEM.

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Current address: Electron Microscopy Sciences, 1560 Industry Road, Hatfield, PA 19440.

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References
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[1]Njisse, J and van Aelst, AC, Scanning 21 (1999) 372–78.10.1002/sca.4950210603
[2]Walther, P, Microsc Microanal 9(4) (2003) 279–85.10.1017/S1431927603030496
[3]Chang, IY-T and Joester, D, Microsc Microanal 21(S3) (2015) 377–78.10.1017/S1431927615002688
[4]Van Dongen, JT et al. , Annals of Botany 91 (2003) 729–27.10.1093/aob/mcg066
[5]Utsumi, Y and Sano, Y, “Cryoplaning Technique for Visualizing the Distribution of Water in Woody Tissues by Cryoscanning Electron Microscopy” in Electron Microscopy: Methods and Protocols: Methods in Molecular Biology, Second Edition, Vol. 369, ed. Kuo, J, Humana Press, Totowa, NJ 2007.
[6]Plumley, WC, Microsc Microanal 20(S3) (2014) 1442–43.10.1017/S1431927614008940
[7]Goodman, SL et al. , Microscopy Today 23(5) (2015) 3037.10.1017/S1551929515000760
[8]Karnovsky, MJ, J Cell Biol 27 (1965) 137–38A.
[9]Murakami, T et al. , Scan Electron Microsc 1983(1) (1983) 235–46.
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Microscopy Today
  • ISSN: 1551-9295
  • EISSN: 2150-3583
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