title: Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.
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.
authors.affiliation: Electron Microscopy Facility, University of Lausanne, Biophore, 1015 Lausanne, Switzerland. Electronic address: Caroline.Kizilyaprak@unil.ch.
authors.forename: Caroline
authors.initials: C
authors.lastname: Kizilyaprak
authors.affiliation: Laboratory of Physics of Living Matter - EPFL, Cubotron - Sciences Physiques UNIL, 1015 Lausanne, Switzerland.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Longo
authors.affiliation: Electron Microscopy Facility, University of Lausanne, Biophore, 1015 Lausanne, Switzerland.
authors.forename: Jean
authors.initials: J
authors.lastname: Daraspe
authors.affiliation: Electron Microscopy Facility, University of Lausanne, Biophore, 1015 Lausanne, Switzerland. Electronic address: Bruno.Humbel@unil.ch.
authors.forename: Bruno M
authors.initials: BM
authors.lastname: Humbel
mesh_terms: D000818:Animals; D004853:Epoxy Resins / Q000737:chemistry*; D021621:Imaging, Three-Dimensional; D008099:Liver / Q000166:cytology; D008810:Mice, Inbred C57BL; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D016613:Plastic Embedding / Q000379:methods
chemical_list: D004853:Epoxy Resins
keywords: Atomic force microscopy (AFM); Durcupan; Epon; Epoxy; Focused ion beam (FIB); Lowicryl; Methacrylate; Radiation damage; Scanning electron microscope (SEM); Shrinkage of resin section; Tomography; Transmission electron microscope (TEM)