258 Histochem Cell Biol (2009) 132:253–262 Fig. 4 A diagram illustrating the vitreous cryo-sectioning technique. a A confocal laser image of Saccharomyces cerevisiae (Dr Maxim Zakhartsev and Doris Petroi, International University Bremen, Ger- many). The cells are rapidly frozen by high pressure freezing (HPF) in copper tubes and then sliced (b) into very thin (50 nm) vitreous cryo- sections. b A relatively long ribbon of vitreous cryosections is held by the operator and pulled away from the diamond knife. The ribbon is placed on an EM grid and imaged by Cryo-EM and -ET. c A medium magniWcation single image showing a 50 nm vitreous slice of a single Saccharomyces cerevisiae cell. Scale bar 2 m. In the corner of the image are holes within the carbon support Wlm (white round circles). have seen indications that compression is rather non-homo- geneous in Wlament bundles (Salje et al. 2009), microtubules (Bouchet-Marquis et al. 2007), desmosomes (Al-Amoudi et al. 2007), and bacterial chemotaxis receptors (Zhang et al. 2004). On the nanomachine level the aVect of these artifacts is uncertain, but could be minimal for rigid, macro- molecular machinery (J. Pierson et al., unpublished). In addition, extreme caution must be taken during image acquisition. Beam induced alterations in the sample are well-known to be a problem, and are often believed to be the rate limiting step for achieving optimal cryo-EM. It is no secret that vitreous sections ‘Xow’ during image acquisi- tion (Sartori Blanc et al. 1998; Sartori, et al. 1996), which is considered by some researchers to be due to local charge accumulation and/or radiochemical processes. Therefore, it is important to ‘learn’ how to read vitreous sections with respect to structural integrity and preservation (Dubochet et al. 2007). A common practice is to acquire a single pro- jection image, using a very low electron dose, before a tilt series is collected, and then compare it to an image taken 123 Subsequent cryo-ET is shown in d, e. d A single 2D projection image from a tilt series. It is diYcult to interpret such images due to the projection problem of overlapping structures, along with a low signal- to-noise ratio and contrast levels. Scale bar 100 nm. e Multiple single projection images can be aligned and reconstructed into a 3D volume (in this case using IMOD (Kremer et al. 1996)). A 5-nm slice from a reconstructed volume. In this image the cellular environment can be visualized much better than in the single projection image (d). The inset displays a selected area (orange-bounded box) from the tomo- graphic reconstruction (e) after the series is complete. If there is no noticeable change, then the structural integrity of the vitreous section has been maintained. If the Wnal image appears ‘smooth’ (with no knife marks and/or crevasses visible) then the ultra-struc- ture of the cell must be analyzed with caution. Map mon- taging helps to reduce the ‘overhead’ electrons used to pinpoint an area of interest before acquiring a tilt series. In order to preserve the vitreous section integrity, the electron beam dose is restricted, which in turn limits the contrast levels and the signal-to-noise ratio. This makes subsequent analysis of such images rather diYcult. The application of tomography of vitreous sections is the future of cellular imaging, combining close-to-native preparation techniques with a 3D view of the vitreous sec- tion. The ultimate goal is to map complex macromolecular machines within 3D slices of large cells and tissue. An example is shown of a sample of Saccharomyces cerevisiae that was immobilized in a copper tube using HPF. The sam- ple was vitreous cryo-sectioned into thin (50 nm) slices and observed using cryo-ET (Fig. 4).