Histochem Cell Biol (2009) 132:253–262 255 Fig. 1 From a whole cell to isolated complexes. a Electron micro- graphs of osmotically shocked S. Xexneri exhibiting the type III secre- tion system protruding through the bacterial envelope (see boxed area). b Gel chromatography of solubilized secretion complexes from the bacterial envelope. c The fraction containing enriched complex is checked by SDS and individual bands identiWed by mass spectrometry. d 2D projection average of 1,500 isolated needle complexes composed of a hollow needle appendage (indicated by a stain-penetrated line along its axis) and a basal part of several rings that traverse the cell envelope. The channel that runs through the needle appendage is 2– 3 nm in diameter indicating that substrates that exit the conduit do so in an unfolded state by using semi-automated computer programs. Particle alignment then modiWes the rotational and translational position of each particle, such that all particles have a comparable orientation and key features will become visible. Since the resolution depends on the accuracy of the particle alignment, there must be suYcient particle feature recognition in a noisy image. Larger complexes are easier to align and subsequently process. A mini- mum particle size of 1,000 kDa is therefore desired for proper alignment (Henderson 1995). The more particles used in the reconstruction, the higher the resolution of the Wnal model. The technique is most powerful when the particles display intrinsic icosahedral (Böttcher et al. 1997), helical (Miyazawa et al. 2003), or crystalline (Henderson et al. 1990) symmetry, that in certain instances such as polyhedrosis virus (Yu et al. 2008), aquaporin-1 (Murata et al. 2000; Engel 2003) and of the bacterial Xagellum (Yonekura et al. 2003) have reached atomic resolution. Asymmetric complexes continue to remain challenging to images using SPA. The ribosome, which is one of the most analyzed low symmetry single particles, is an excep- tion. It has served for many years as test specimen in the development of many single particle techniques (Stark 2002). Although the resolution is lower for such asymmet- ric particles, a quantitative description, on the quaternary level, is nevertheless possible. Visualizing nanomachines in a cellular context A drawback with the single particle technique is that the nanomachines may undergo changes during isolation, such as loss of labile components, or certain conforma- tional states may be favored. Furthermore, there are many macromolecular assemblies associated with membranes, which should be studied without extraction, in a cellular context. Specimen preservation is one of the most critical steps in the entire process of native cellular imaging. Cellular water in liquid form is incompatible with the vacuum of the EM. Water is the most abundant cellular constituent and there- fore important for preserving cellular ultra-structure. Cur- rently the only way to Wx cellular constituents without introducing signiWcant structural alterations is by cryo- Wxation. To be successful, the freezing process has to be ultra-rapid in order to avoid ice crystal formation, which damages cellular material and can hinder imaging. There are currently two common methods employed; plunge freezing into liquid ethane (Dubochet et al. 1988) and high pressure freezing (HPF, Studer et al. 2001). Cryo-Wxation The plunge freezing technique is suitable for samples with limited thickness [i.e., bacteria, isolated cellular organelles 123