Vitreous Cryosectioning
Phone: +49 - 89 - 8578 2646
Alumni:
Dr. Christian Hoffmann
Dr. Manuela Gruska
Dr. Valerio Matias
Dr. Christoph Hagen
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.
In principle, vitrified cryosectioning of high-pressure frozen (HPF) specimens offers the possibility to perform tomography on thin sectioned objects. However, the method is infamous for the mechanical distortions it causes to the specimen. The first is x-y compression of more than 30% and a corresponding increase in thickness. The second is the production of surface cracks (crevasses) which become more severe with increasing thickness of the sections. Therefore, the usefulness of vitrified sections for 3D-work is limited. In addition the range of specimens suited to HPF for vitreous sectioning is still very limited. The best are suspensions of cells, such as cultured mammalian cells (2), algae (3), bacteria (6), or yeast (7). Larger high-pressure frozen specimen (e. g., Drosophila embryos) can yield excellent results in cryosubstitution.
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 and cracks, allowing quantitative measurements of subcellular dimensions, but there is no serial sectioning capability.
A promising application for cryosectioning lies in the immunocytochemical localization of proteins in either high-pressure frozen or chemically fixed specimens (Tokuyasu). This method at least avoids dehydration and embedding artefacts and has yielded useful results.
Conventional sectioning of plastic-embedded material is still a method for investigation of most bulk specimen, such as animal tissue.
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].
Two ultramicrotomes (Leica FCS and FC6) for cryosectioning, one ultramicrotome (Leica Ultracut) for conventional plastic sectioning, with a range of diamond knives
Plunge-freezing and high-pressure freezing equipment (Leica Empact 2)
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
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Dr. Alexander Rigort
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