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MPI für Biochemie  

Molecular Structural Biology
Wolfgang Baumeister

Vitreous Cryosectioning

 

Members

Dr. Thomas Keil

Phone: +49 - 89 - 8578 2646

Thin ice for electron microscopy

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We investigate methods suitable for thinning ice-embedded cells and tissues to enable 3D structural studies by cryo electron tomography. Here, ‘suitable’ means that, during thinning and microscopy, the specimen must not be allowed to warm to more than -137°C. At this temperature, amorphous ice undergoes a transition to a crystalline and thus structure-damaging phase.

 

Low-temperature ultramicrotomy using a diamond knife is the established way to obtain thin slices of ice-embedded cells suitable for cryo- electron tomography. This technique offers the possibility to perform tomography on serially sectioned objects; however, it is infamous for the mechanical distortions that it causes to the specimen, the most serious of which is x-y compression of more than 30% and a corresponding increase in thickness. Other distortions do not affect the entire section volume. An alternative method uses a focussed ion beam to ablate a specimen, layer by layer, until it is suitably thin for tomography. Preliminary indications are that the specimen remains vitreous after milling if the relevant parameters are set carefully. Unlike knife-based sectioning, ion beam milling does not induce compression, allowing quantitative measurements of subcellular dimensions, but there is no serial sectioning capability.

 

Together with colleagues from the 3DEM Network of Excellence, we have established ways of applying colloidal, electron-dense particles to thinned vitreous specimens at cryogenic temperatures, greatly simplifying the alignment of tomographic tilt series without introducing foreign particles into cells [1, 2]. We also take advantage of the relatively uniform thickness of sectioned ice and the slowed bleaching properties of fluorophores at low temperature to record complementary fluorescence images of vitrified, sectioned cells [3-5].

Key instrumentation

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Two cryo-ultramicrotomes with a range of diamond knives

Plunge-freezing and high-pressure freezing equipment

Cryo-DualBeam microscope (focussed ion beam + field emission scanning electron microscope)

Cryo light microscope (own stage design and construction)

Range of cryo electron microscopes for data acquisition

Publications

  1. Masich, S., et al., A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography. Journal of Structural Biology, 2006. 156(3): p. 461-468.
  2. Gruska, M., et al., Electron tomography of vitreous sections from cultured mammalian cells. Journal of Structural Biology, 2008. 161(3): p. 384-392.
  3. Leis, A., et al., Cryo-electron tomography and fluorescence microscopy of unicellular algae in vitreous cryosections. Microscopy and Microanalysis, 2005. 11(Suppl. 2): p. 330CD.
  4. Sartori, A., et al., Correlation microscopy: Bridging the gap between light- and cryo-electron microscopy. Microscopy & Microanalysis, 2005. 11(2): p. 16-17.
  5. Sartori, A., et al., Correlative microscopy: Bridging the gap between fluorescence light microscopy and cryo-electron tomography. Journal of Structural Biology, 2007. 160(2): p. 135-145.