Electron tomography of vitreous sections from cultured mammalian cells

Manuela Gruska, Ohad Medalia, Wolfgang Baumeister, Andrew Leis

Research output: Contribution to journalArticlepeer-review

72 Scopus citations

Abstract

Cryo-electron tomography of appropriately thin, frozen-hydrated biological specimens has excellent potential for investigating the 3D macromolecular architecture of eukaryotic cells and tissues. Since cardiomyocytes are too thick to be visualised in an intact state, we grew immortalised cell line HL-1 to sub-confluency and harvested the cells by enzymatic detachment prior to hyperbaric freezing, ultramicrotomy, and tomography. We improved the efficiency of tomographic acquisition from vitreous cryosections by implementing two new features: (1) fluorescence microscopy at cryogenic temperatures to search for features of interest without expending any of the tolerable electron dose on secondary (non-imaging) tasks, and (2) the use of colloidal gold as fiducial markers. Vital fluorescent staining and subsequent cryo-fluorescence microscopy of vitreous sections were used to localise mitochondria lying in positions suitable for acquiring tilt series, taking into account section flatness, presence of contamination and proximity to grid bars. To provide a simple and robust means of aligning tomograms, we developed a universally applicable protocol for depositing colloidal gold onto vitreous sections, analogous to the method for applying quantum dots described by Masich et al. [Masich, S., Östberg, T., Norlén, L., Shupliakov, O., Daneholt, B., 2006. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography. J. Struct. Biol. 156, 461-468]. Tomograms of thin sections (nominal thickness 65-85 nm) of cardiac mitochondria revealed the interconnectivity of cristae and junctions with the inner mitochondrial membrane. In some cases, ATP synthases could be identified without ambiguity. These findings confirm the feasibility of investigating the structural biology of mammalian cells in three dimensions and at a resolution of 6-8 nm.

Original languageEnglish
Pages (from-to)384-392
Number of pages9
JournalJournal of Structural Biology
Volume161
Issue number3
DOIs
StatePublished - 1 Mar 2008

Keywords

  • ATP synthase FF dimers
  • Colloidal gold
  • Correlative microscopy
  • Cryo-electron tomography
  • Cryo-fluorescence microscopy
  • Cryosections
  • HL-1 cardiomyocytes
  • Mammalian mitochondria
  • P19 embryonic stem cells
  • Vitreous sections

ASJC Scopus subject areas

  • Structural Biology

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