title: Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.
abstract: Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.
authors.affiliation: Electron Microscopy Facility, University of Lausanne, Biophore, 1015 Lausanne, Switzerland. Electronic address: Celine.Loussert@unil.ch.
authors.forename: Celine
authors.initials: C
authors.lastname: Loussert Fonta
authors.affiliation: CSIRO, Australian Animal Health Laboratory, Private Bag 24, Geelong 3220, Australia.
authors.forename: Andrew
authors.initials: A
authors.lastname: Leis
authors.affiliation: FEI Company, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands.
authors.forename: Cliff
authors.initials: C
authors.lastname: Mathisen
authors.affiliation: Department of Fundamental Neurosciences, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland.
authors.forename: David S
authors.initials: DS
authors.lastname: Bouvier
authors.affiliation: Electron Microscopy Facility, University of Lausanne, Biophore, 1015 Lausanne, Switzerland.
authors.forename: Willy
authors.initials: W
authors.lastname: Blanchard
authors.affiliation: Department of Fundamental Neurosciences, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Volterra
authors.affiliation: FEI Company, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands.
authors.forename: Ben
authors.initials: B
authors.lastname: Lich
authors.affiliation: Electron Microscopy Facility, University of Lausanne, Biophore, 1015 Lausanne, Switzerland.
authors.forename: Bruno M
authors.initials: BM
authors.lastname: Humbel
mesh_terms: D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D016616:Cryoultramicrotomy / Q000379:methods*; D007150:Immunohistochemistry; D051379:Mice; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D008856:Microscopy, Fluorescence
keywords: Acute brain slice; Astrocyte; Correlative light–electron microscopy; Green fluorescent protein (GFP); Immunolabelling; Scanning transmission electron microscopy (STEM); Tissue; Tokuyasu cryo-sectioning