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Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

Authors
  • Kizilyaprak, Caroline
  • Longo, Giovanni
  • Daraspe, Jean
  • Humbel, Bruno M
Type
Published Article
Journal
Journal of Structural Biology
Publisher
Elsevier BV
Publication Date
Feb 01, 2015
Volume
189
Issue
2
Pages
135–146
Identifiers
DOI: 10.1016/j.jsb.2014.10.009
PMID: 25433274
Source
Medline
Keywords
License
Unknown

Abstract

In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼ 500 nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples.

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