Cryo-electron tomography (cryo-ET) gives scientists the power to achieve nanometer scale, 3D imaging of unperturbed cellular environments. Cryo-ET has been successfully used to resolve previously hidden structural details of multi protein complexes and their distribution within cells. Further optimization and combination of cryo-ET with other analytical techniques promises unprecedented insight into the sophisticated protein interactions that drive cellular activity.
Recent advances in sample preparation and analysis have transformed electron microscopy (EM) into a powerful tool for characterizing the molecular organization inside cells. Pairing the novel method of cryo-electron tomography (cryo-ET) with other existing techniques for biological imaging and biochemical analysis could revolutionize the way scientists understand the complex workings of the proteome1,2.
Every cell contains within it a diverse array of highly modular multi-component ‘machines’ composed mostly of proteins, the concerted action of which drives essential cellular processes such as gene expression, cell division and metabolic activity. Some of these complexes are stable enough to purify and characterize with atomic-level detail via techniques such as X-ray crystallography, but many others are based on brief and weak interactions3.
Although experimental techniques exist that help scientists deduce the composition and function of such complexes, a preferred solution would be to apply high-resolution imaging techniques to visualize them in their cellular context without disrupting the physiological environment4,5. Such an approach would form the basis of ‘visual proteomics’, in which the various proteins within a cell or another biological sample can be directly observed in their natural habitat rather than in a heavily manipulated experimental preparation.
Biological imaging is undergoing a renaissance. So-called super-resolution light-microscopy (LM) techniques now allow investigators to observe dynamic processes in living cells at resolutions of less than 50 nm. However, these techniques are presently incapable of providing true molecular-scale detail and can only highlight structural features carrying a fluorescent label, while the context remains unclear. EM has continued to evolve as a potent and complementary technique, delivering unparalleled levels of detail in frozen ‘snapshots’ of cells.
Electron microscopy samples were traditionally prepared with toxic chemical fixation and staining agents, and water had to be removed before transferring the samples into the vacuum of the electron microscope. Such treatments tend to disrupt the arrangement of cellular components and yield misleading visual artefacts6.
Frozen in time
ET is a variant of EM in which samples are rotated and imaged from various angles, yielding multiple projection images that can be reconstructed into three-dimensional pictures of cells, organelles and other structures. Instead of removing any liquid water, samples are rapidly frozen such that they become embedded in ‘vitreous’ ice. In this medium, as with glass, water molecules are arranged randomly rather than in the crystalline form that can damage biological samples.
The resulting technique of cryo-ET thus avoids the artefacts associated with conventional EM, and delivers highly detailed and spatially accurate reconstructions of the intact cellular environment at nanometer resolution. However, the absence of staining agents means that scientists must contend with reduced contrast, making it more difficult to distinguish structures of interest against the background of the cellular environment. This is exacerbated by the fact that ice-embedded samples are vulnerable to damage from prolonged exposure to the electron beam, meaning that researchers must minimize exposure, which results in a low signal-to-noise ratio.
In order to maximize the quality of the information that can be obtained from these tomograms without compromising sample integrity, scientists have paired clever strategies for specimen preparation with sophisticated computer algorithms for data processing and analysis. This has allowed scientists to sucessfully employ cryo-ET to visualize a number of complicated cellular systems including the following: the nuclear-pore complex, which is the ‘gatekeeper’ to the nucleus; the presynaptic terminals, from which neurons issue stimulatory and inhibitory signals to their neighbours; and strings of polyribosomes, which are the assembly lines that manage the production of protein from RNA (Fig. 1). For smaller cells, such as bacteria, it even becomes possible to characterize the distribution of populations of large multi-protein complexes within the full cellular volume, and early efforts at performing organism-wide visual proteomics have been described for the human pathogens Leptospira interrogans and Mycobacterium pneumoniae9.10 .
Visualizing the future
Cryo-ET is still an emerging technology and is not yet in widespread use, but the near future should see considerable improvements that will greatly expand the power of this tool. For example, although the area that can be effectively visualized with cryo-ET is suitable for examining many bacteria in their entirety, it remains too small to cover a human or mouse cell. This size limitation can be overcome by so-called ‘correlative LM– EM’ approaches, in which LM is used to survey samples at low resolution in order to identify sites of interest for high-resolution analysis via cryo-ET. Improved methods of dissection will also be necessary to ensure that researchers can get easy access to target zones within a large cellular landscape — for example, using a focused ion beam to ‘shave’ a frozen sample precisely down to an appropriate depth for imaging.
More precise micromachining methods could also lead to more comprehensive, top-down approaches for understanding cellular machinery. For example, a cellular territory could be characterized at a structural level via cryo-ET, and then carefully excised for analysis using mass spectrometry or related techniques that allow scientists to reconstruct comprehensive inventories of the individual proteins within a given sample volume. This could, in turn, contribute to the generation of molecular atlases containing valuable details about how different cell types respond to external signals, damage or other environmental triggers.
Of course, these data will also require more sophisticated software tools; data analysis remains tedious and time-consuming, and still depends heavily on human oversight. Ideally, future algorithms will be capable of automatic image interpretation with the capacity to distinguish individual objects or patterns within complex environments.
Given the progress to date, it seems reasonable to expect that, in the near future, cryo-ET will achieve levels of resolution and sophistication that allow scientists to accurately reconstruct the complex behaviours and dynamics of a diverse array of multi-component molecular systems — as well as the interplay between them — and thereby gain unprecedented insight into the ‘molecular sociology’ of the cell.
Humans are a highly visual species and, in the wake of new imaging techniques, the life sciences are now entering another visual phase. Cryo-electron tomography fills a critical gap between techniques with atomic resolution and light microscopy, bridging the divide between molecules and cellular structural studies. It provides unprecedented insights into the molecular reality of cells (Leis, A. et al. Trends Biochem. Sci. 34, 60–70, 2008).