Trends in Biochemical Sciences
ReviewVisualizing cells at the nanoscale
Section snippets
A role for cryo- electron tomography in molecular cell biology
Cellular processes rely on the concerted action of macromolecular ensembles – functional modules – and on networks of transiently interacting components. Proteomics has greatly advanced our knowledge of the inventories of cells and organelles but conjecture remains regarding the spatio-temporal organization of proteomic networks. On a systems level, our current understanding of interaction networks is rather limited, mainly because we currently lack the experimental tools necessary for the
Accurate preservation of cellular architecture
The key prerequisite for mapping cellular landscapes in situ is maintaining the delicate molecular and supramolecular architecture of the cell during sample preparation. To observe cells in the electron microscope, water must be removed and replaced by a non-volatile medium or frozen. The removal or substitution of water inevitably results in the redistribution of ‘soluble’ cytoplasmic components and precipitation on membranes and the cytoskeleton. The portrayal of such artefacts led to the
Principles and limitations of cellular tomography
Electron tomography, like other tomography modalities, relies on the principle of recording images from different viewing angles, aligning the resulting ‘projections’ to a common coordinate system and recombining them computationally to form a 3D image volume (Box 1). Electrons or X-rays can be used to perform cellular tomography and both modalities can exploit cryogenic conditions to minimize radiation damage [17]. Cryo- ET can be used to visualize many prokaryotic cells directly [18];
Preservation by vitrification
In relation to electron microscopy of cells and tissues, it has been stated that ‘obtaining optimal results is often a compromise between maximum preservation and the clear visualization of structures’ [39]; however, if the aim is to visualize molecular structure and supramolecular architecture, we must not allow structural preservation to be compromised. At present, there are two convenient ways of preserving cells in amorphous ice before cryo- ET: (i) ‘plunge freezing’ in a secondary cryogen
Correlative imaging with spatial and temporal resolution
The judicious use of multiple imaging techniques can provide complementary information concerning cell structure and function (Figure 1). These techniques should not only span several orders of magnitude in spatial resolution but they should also enable one to monitor cellular processes and capture them at crucial points in time. Vitrification provides a ‘snapshot’ with a temporal resolution in the range of milliseconds. An integrated approach must, therefore, be developed to observe specific
Ultramicrotomy: the ‘Achilles heel’ of electron tomography
Mechanically generated slices of vitrified cells inevitably suffer from mechanical distortions. Tomograms of cryosections are helpful in understanding how these artefacts arise and they provide clues as to how artefacts can be minimized. So-called ‘crevasses’ [58], deformations that are especially apparent in thick vitreous sections (>100 nm), dominate superficial regions of the section surface distal to the knife surface and diminish with depth. Although thinner sections (25–75 nm) are currently
Molecular interpretation of tomograms
Cryo- electron tomograms of organelles and cells contain vast amounts of information that extends beyond cellular ultrastructure. Essentially, they are 3D representations of the entire proteome and they are snapshots of the interaction networks underlying cellular functions. However, retrieving this information is not a trivial task because the signal-to-noise ratio of the tomograms is low and individual macromolecules are difficult to recognize in an environment that is so crowded that they
Concluding remarks and future perspectives
The human being is a highly visual-centric species and, in the wake of new technologies, the life sciences are entering another highly visual phase [73]. Super-resolution light microscopes have surpassed the ‘diffraction barrier’, electron microscopy has left behind the artefact-stricken methods developed over the past 50 years in favour of rapid freezing and direct visualization and X-ray microscopy is beginning to live up to expectations. Cryo- ET has the unique potential to bridge the divide
Acknowledgements
Work in our laboratory was supported by the 3D-EM Network of Excellence within the Sixth Framework Programme financed by the European Commission. We thank Julio Ortiz, Martin Beck, Emmanuel Burghard and Günther Gerisch for contributing portions of the figures.
Glossary
- Alignment
- prior to reconstruction (Box 1), individual projection images are mutually positioned in a tomographic tilt-series to account for physical shifts.
- Amorphous ice
- also known as vitreous water; any phase of non-crystalline ice formed by a sufficiently rapid cooling rate, which can be verified by electron diffraction.
- Cryogen
- typically a liquid that boils below −160 °C, used for cooling cells to arbitrarily low temperatures; classed either as primary cryogens (e.g. liquid nitrogen) or secondary
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