title: The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications.
abstract: The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.
authors.affiliation: National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, 1070 Basic Science Building MC 0608, 9500 Gilman Drive, La Jolla, CA 92093-0608, USA. gsosinsky@ucsd.edu
authors.forename: Gina E
authors.initials: GE
authors.lastname: Sosinsky
authors.forename: John
authors.initials: J
authors.lastname: Crum
authors.forename: Ying Z
authors.initials: YZ
authors.lastname: Jones
authors.forename: Jason
authors.initials: J
authors.lastname: Lanman
authors.forename: Benjamin
authors.initials: B
authors.lastname: Smarr
authors.forename: Masako
authors.initials: M
authors.lastname: Terada
authors.forename: Maryann E
authors.initials: ME
authors.lastname: Martone
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.forename: John E
authors.initials: JE
authors.lastname: Johnson
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D004330:Drosophila / Q000648:ultrastructure* / Q000821:virology; D009474:Neurons / Q000648:ultrastructure*; D011312:Pressure; D016707:Tissue Fixation / Q000379:methods*; D014054:Tomography / Q000379:methods*; D053586:Virus Internalization