260 The future of cryo-electron tomography The current, rather conservative, approximation of the resolution limit of 4–5 nm is expected to improve as the need for high-resolution structural information steadily increases. The electron source is a suitable starting point for improvement. We have witnessed a dramatic increase in spatial coherence of electrons using a Weld emission electron gun (FEG) rather than a LaB6 or tungsten Wla- ment. As the FEG improves, the induced specimen dam- age will be further minimized and the resolution may improve. Contrast is generated diVerently with unWxed, unstained material. The objective lens is defocused creating phase contrast. By defocusing the objective lens, the contrast transfer function is altered and for high resolution studies it will need to be accurately assessed through the volume of single projection images, which is not trivial compared to single projection images. A comparison between CTF corrected and uncorrected can be seen in chromosome arrangement within mitotic HeLa cell sections (Eltsov et al. 2008). Electron detectors for image collection are also becom- ing more sophisticated. The problem of electrons backscat- tering from the Wberoptic plate into the scintillating layer of a charge couple device in current detectors can be partially solved by direct detection of electrons (a complementary metal-oxide-semiconductor) rather than relying on a scintil- lating layer (Jin et al. 2008). Remote microscopy control allows users from around the world to operate and control microscopes in a completely diVerent part of world. We have seen that the loading of cryo samples has now become completely automatic thanks to the cryo-autoloader, which is an integral part of the Titan Krios (FEI Company). One million particles taken from several EM grids under cryo- genic conditions can be imaged and processed automati- cally in 1 week (H. Stark, personal communication). This trend should not be limited to imaging but also to the sam- ple preparation. In the past two decades, scientiWc challenges in physics, chemistry, and molecular cell biology have moved toward the molecular, nanometer-scale domain. Nanoscience, once only a discipline of theoretical subjects, has now been established as a vibrant multidisciplinary area of research and development. For EM labs to participate fully in this Weld, it will be necessary to rationalize EM resources. In the Netherlands for example, about 25 EMs are dispersed throughout the country. Some have old instruments and low scientiWc impact, while their maintenance (service) costs are high. None have the resources needed to initiate a Dutch EM cen- ter as part of the much-needed network of major European centers outlined in the strategic roadmap of the European 123 Histochem Cell Biol (2009) 132:253–262 Strategy Forum for Research Infrastructure (ESFRI). A pre- requisite to realizing these potential centers, however, is a source of funds for such a major facility and to merge resources and personal. National cryo-EM centers will be optimally suited to become centers of excellence in speciWc areas of molecular life science and health and there are sci- entists in many countries with excellent track records in their respective EM-related Welds. With the combined eVort, expertise and training as was shown within the EU 3D-EM network, several centers should emerge with bril- liant junior and senior principle investigators that should have the right scientiWc setting for acquiring suYcient research grants to make it self sustainable with the help of bright young scientists. We are working toward generating such a center. For more information please visit http:// www.necen.nl. Summary The cell is composed of a highly organized network of macromolecular machines addressing the demanding requirements of a living cell. Imaging these ‘nanoma- chines’ in a cellular, close-to-native state is not a trivial task. Isolation techniques will continue to be essential in solving the structures of these types of protein complexes by the ‘divide and conquer’ method (McIntosh 2007). For analysis of these nanomachines in their normal environ- ment, rapidly freezing the cell, and cellular elements, in a vitreous layer of water is currently the only technique that retains a physiological state of the organism or cells before EM imaging. As we have seen (Nicastro et al. 2006), unmatched structural information can be obtained. Using vitreous cryo-sections, Cryo-ET can be extended to any cell and almost all tissue. Yet, one major limitation is the proper vitriWcation under physiological conditions and the locali- zation of each macromolecular complex, along with their binding partners, throughout the thickness of the vitreous cryo-section. As the Weld of cellular imaging of nanoma- chines continues to evolve, we are certain to gain invalu- able cell biological insights. We are also likely to see many nanotechnology applications that utilize these elegant cellu- lar machines. Acknowledgments We thank Shoaib Amini for assistance with Figs. 4 and 5. We also thank members of the Peters lab and members of the Netherlands Centre for Nanoscopy (http://www.necen.nl) and Helmut Gnaegi from Diatome for stimulating discussions. This work was performed with support oV the Sixth Research Framework Pro- gram of the European Union, Project ImmunoPrion (Food-023144), the NIMIC consortium (Nano-IMaging under Industrial Conditions http://www.realnano.nl) and Aeras Global TB Vaccine Foundation which has received a grant for this project from the Netherlands Direc- torate-General of Development Cooperation (DGIS) Dutch Ministry of Foreign AVairs.