Histochem Cell Biol (2009) 132:253–262 DOI 10.1007/s00418-009-0622-0 R E V I E W Toward visualization of nanomachines in their native cellular environment Jason Pierson · Musa Sani · Cveta Tomova · Susan Godsave · Peter J. Peters Accepted: 7 July 2009 / Published online: 1 August 2009 © The Author(s) 2009. This article is published with open access at Springerlink.com Abstract The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanoma- chines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional Wxatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecu- lar resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or Wtted into the lower resolution particle density maps to create a macromolecular atlas of the cell under nor- mal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous Robert Feulgen Lecture 2009 presented at the 51st symposium of the Society for Histochemistry in Stubai, Austria, October 7–10, 2009. J. Pierson · M. Sani · C. Tomova · S. Godsave · P. J. Peters (&) Division of Cell Biology, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital (NKI-AVL), Plesmanlaan 121 B6, 1066 CX Amsterdam, The Netherlands e-mail: p.peters@nki.nl URL: www.nki.nl/research/peters fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological consider- ations for visualizing nanomachines in a close-to-physio- logical, cellular context. EM is in a renaissance, and further innovations and training in this Weld should be fully supported. Keywords Cellular nanomachines · Single particle analysis · Cryo-electron tomography · Vitreous cryo-sectioning Introduction Proteins perform key roles in the majority of cellular func- tions. They also serve as building blocks for larger heterogeneous macromolecular assemblies, or biological nanomachines, which are organized into cellular ‘modules’ (Alberts 1998). As the name implies, a biological machine is analogous to the man-made version. Both are made of individual components that function in a coordinated fash- ion to perform speciWc functions. In nature, if any single structural component of a nanomachine malfunctions the result is often a disease. Discerning the ultra-structure of a biological machine and its components within the cell can contribute to an understanding of how it functions both nor- mally, and in disease. Little is known, however, about the details of how these macromolecular machines are spatially organized within a cellular environment. X-ray crystallography and NMR approaches have been used to complement genetics, biochemistry, and proteomics to characterize the overall structural organization of indi- vidual protein complexes. Most macromolecules, however, cannot be crystallized, or when crystallized, they diVract poorly, as only large, well-ordered crystals diVract. In the 123