Histochem Cell Biol (2009) 132:253–262 259 Fig. 5 Charting macromolecular machines within a vitreous cryo-sec- tion. a A selected area (orange-bounded box in Fig. 4d) from a tomo- graphic reconstruction. b, c The volume has been surface rendered in order to visualize the cellular organization of macromolecular com- plexes within a thin vitreous cryo-section from a whole Saccharomyces cerevisiae cell Within the volume of the reconstruction, individual macromolecular complexes can be observed (Fig. 5). Based on their size and distribution we assume that these are native 80S ribosomes in various conformational states, which can also be associated with a variety of pro- tein complexes that aid in protein translation. The true identity, however, remains subjective until a suitable labeling or correlative approach is developed for the vitre- ous cryo-sectioning. The future of charting nanomachinery in a native cellular context We have witnessed light microscopy breaking the diVrac- tion barrier, and in the future the spatial resolution will only improve, with the development of super-resolution light microscopes. Recently it became clear that the green Xuo- rescent protein (GFP) tag has a 10–50 times better signal- to-noise ratio at cryogenic conditions because of reduced photo-bleaching (Sartori et al. 2007). In combination with EM, correlative cryo-LM–EM oVers a promising opportu- nity to investigate cellular organization (Sartori et al. 2007; Schwartz et al. 2007; Plitzko et al. 2009). Rather than using a single clonable Xuorescent (GFP) tagged construct widely used in light microsocopy, a heavier circular string of Wve GFP’s in tandem could be constructed that would be visible in the rather noisy and low contrast cryo-ET images. In addition, the GFP could be coupled to ferritin or another analog electron dense metal- lothionein molecule to provide a label for cryo approaches (Mercogliano and DeRosier 2007). High-resolution structures produced by X-ray crystal- lography or NMR can be Wtted or docked into lower resolution EM images to create a multi-resolution map of the protein structure. Such bridging techniques will be instrumental in providing a more complete understanding of the structure and function of molecular complexes than is possible with a stand-alone technique. Recently, volumetric averaging, which is based on the single particle averaging techniques presented, was applied to isolated organelles (Nicastro et al. 2006) and to cellular protein complexes involving cadherins in epidermal des- mosomes of native skin (Al-Amoudi et al. 2007). In the lat- ter case, a high-resolution X-ray crystal structure was placed into the lower resolution cadherin EM map, which resulted in a multi-resolution map of the cadherin in native epidermal desmosomes. In our own lab, one aim is to further our knowledge on the mechanism of phagocytosis of Mycobacterium tubercu- losis in human cells. Contrary to the previously accepted view, we have shown using cryo-immunogold EM that the mycobacteria can translocate from the conWnes of the phagosomal membranes into the cytosol, where they repli- cate more rapidly and cause cell death (van der Wel et al. 2007). We are now using SPA to investigate the type VII secretion system in isolated bacteria and ultimately we aim to image mycobacteria in sections of HPF frozen human cells, and to gain an understanding of the mechanism for type VII-mediated translocation. The SPA, X-ray and NMR data can then be docked into the images from vitreous sec- tions to construct a macromolecular map of the tubercle bacillus within the host cell. ‘Cutting edge’ technology The only way to look inside large cells and tissue, at a close-to-native preservation, is by HPF and vitreous sec- tioning for cryo-EM. The diamond knife and accessory tools used for cryo-sectioning are improving, which will tremendously improve both the quality of the vitreous sec- tions and subsequently the quality of the imaging. We fore- see that in the near future we will use robotics for section manipulation to make handling even more convenient. An alternative approach to mechanical sectioning is based on focused ion beam milling (Marko et al. 2007). A futuristic ‘nano-knife’ has also been proposed (Singh et al. 2009) constructed from multi-walled carbon nanotubes. In the future these techniques may be able to reduce the distor- tions currently caused by mechanical vitreous sectioning with a diamond knife, but at the current state are rather technically demanding and in development. 123