254 case of NMR, the size of the protein complex is limited to approximately < 100 kDa (Wider and Wüthrich 1999). In order to study biological machines where they normally reside, and not as isolated entities, then more suitable imag- ing techniques, such as electron microscopy (EM) and elec- tron tomography (ET), need to be adapted. Transmission EM is one of the most powerful techniques for ultra-structural studies of the cell and its constituents (McIntosh 2001). When performed on sections of Wxed cells and tissues, it has been invaluable in establishing a picture of the subcellular arrangement of organelles and determining the localization of gene products with immuno- gold labeling techniques. As an example, recently, the route of tubercle bacillus infection was demonstrated using immunogold labeling on aldehyde Wxed cryo-sections and our Wndings revealed a pathway that is against the current textbook dogma (van der Wel et al. 2007). Nevertheless, this technique makes use of Wxed, dehydrated and heavy metal-stained material, and artifacts at the macromolecular level may be introduced at all stages (Peters and Pierson 2008). Recently, there has been an important focus on opti- mal sample preservation for high-resolution structural stud- ies of macromolecules in situ. This quest to Wnd the true relation between molecules, linked into supramolecular complexes and assembled into an intricate network of cel- lular compartments, is revolutionizing modern transmission EM. The catalyst for this revolution has been cryo-ET (Dubochet et al. 1988; Lucic et al. 2008). ET is used to gen- erate 3D maps from a series of 2D transmission EM images. The specimen is rotated with respect to the imaging source; in this case, electrons and a series of projection images are recorded over a limited angular range (usually ¡70° to +70°). Cryo-ET is based on a freezing technique that captures the cellular water in an amorphous (glass-like) layer in which all cellular components are embedded. This process is known as vitriWcation, which is achieved by the ultra- rapid freezing of a thin biological sample, circumventing all harsh chemical Wxatives and heavy metal stain. Here we analyze diVerent EM imaging procedures that have the ultimate goal of visualizing macromolecular ‘machines’ in the most natural environment currently possi- ble. Using a variety of imaging techniques, structural infor- mation can be extracted at diVerent resolution levels. This information can be combined to create a multi-resolution density map of the individual macromolecular complexes; eventually leading to a catalog of all biological nanomachines that can be used to search in 3D volumes (Bohm et al. 2000) of cells, and to determine the spatial organization of individual complexes within a cellular environment (Nickell et al. 2006). In addition, we give insight on what will be important for the future to achieve this goal of mapping macromolecular machines within a cellular context. 123 Histochem Cell Biol (2009) 132:253–262 Single particle analysis Electron microscopy has provided important structural information about protein complexes essential for describ- ing their functional organization. Single particle analysis (SPA) has been used to determine the structure of a variety of macromolecules and biological nanomachines. This involves the analysis of images from large numbers of puri- Wed, macromolecular particles that have been placed on an EM grid. These images are used to build up a 3D model of the complex. Sample preparation For SPA, a convenient isolation approach is to ‘Wsh’ the nanomachines out from their cellular environment using a tag that targets a single component of the complex, most importantly often in a single step (Kelly et al. 2008). Nega- tive staining is often the Wrst step to check for quality and suitability. The sample is embedded in heavy metal salts, such as uranyl acetate or phosphotungstic acid, providing a contrasting agent for the weakly electron-scattering biologi- cal molecules. This staining has been part of the most com- monly used EM preparation methods since its introduction 50 years ago (Brenner and Horne 1959). An example can be seen in Fig. 1 that shows a negatively stained S. Xexneri needle appendage. The averaged projection view shows the basal part of the needle complex, composed of several rings that traverse the cell envelope. The heavy metal stain can, however, create a cast around the surface of the specimen. Moreover, dehydration of the specimen can potentially alter Wne structural details. It also imposes a resolution limit of 10–20 Å (Amos et al. 1982; Sani et al. 2007). These limita- tions have been overcome by cryo-Wxation, or embedding of the specimen in vitreous water (Dubochet et al. 1988). Image analysis Irrespective of the sample preparation method, individual particles must be aligned and averaged to improve the signal. For cryo-preparation methods the averaged particle can con- tain information to atomic resolutions (Henderson 1995; van Heel et al. 2000), but suVer from limited contrast. The imag- ing electron dose is limited (low-dose imaging) to 10–20 electrons/Å2 (Glaeser and Taylor 1978; Dubochet et al. 1988) to reduce the number of inelastically scattered elec- trons (Stark et al. 1996) that often lead to radiation damage. To overcome this problem, single particle image enhance- ment programs have been developed (van Heel et al. 1996; Frank et al. 1996) to average large numbers of copies of sin- gle particles and improve the signal-to-noise ratio. The Wrst step in the averaging procedure is to identify individual particles within the micrograph manually or