Electron Tomography is a relatively new method that allows the investigator to study anatomical structures in thicker specimens than traditional TEM, and to visualize structures that may lie at random angles to the angle of sectioning. Data is collected from a specialized transmission electron microscope while the semi-thin section is viewed at a series of tilt angles; thus physical relationships can be compared throughout a substantial volume of tissue. Tilt series can be collected along both the X- and Y-axes to maximize the fineness of the dataset. A computed “tomogram” is derived from this series of TEM images, after which several software packages are available for image segmentation.

This technique is complementary to X-ray crystallography or NMR in the study of single molecules, and is also becoming useful in the study of whole cells or organelles. Below we show one example of a wild type nematode tail that was compared along two tilt axes and in a “focus-through” series in one thick section to illustrate the relationships between the anal dilator muscle, the rectal epithelium and the rectal cuticle. Using tomography, one can quickly see subcellular details of the basal lamina, and of filamentous structures spanning between these cell types, as well as the cytoskeletal elements within the muscle cell itself. Hall and Leslie Gunther used an FEI Technai 20 electron microscope to produce the images shown here. The sample was originally prepared by high pressure freezing and freeze substitution prior to sectioning. All necessary equipment for this technique is available here.

Electron Tomography Movie Clips - click on picture to open movie

All movies show the specializations where the anal dilator muscle meets the rectal epithelium and anchors to the roof of the rectum. Use the labelled image below as a reference for identifying specific structures.

Tilting through section along X-axis MOVIE 1
Tilting through section along Y-axis MOVIE 2
Focusing through section along Z-axis MOVIE 3
See the following papers for additional details on this technique: Miao, L., Yi, K., Mackey, J.M. and Roberts, T.M. (2006) Reconstitution in vitro of MSP-based filopodium extension in nematode sperm. Cell Motility and the Cytoskeleton, 64: 235-247. Abstract Article Miao, L., Vanderlinde, O., Liu, J., Grant, R.P., Wouterse, A., Shimabukuro, K., Philipse, A., Stewart, M. and Roberts, T.M. (2008) The role of filament-packing dynamics in powering amoeboid cell motility. Proc Natl Acad Sci 105: 5390-5. Abstract Article Müller-Reichert, T., Srayko, M., Hyman, A., O'Toole, E.T. and McDonald, K. (2007) Correlative light and electron microscopy of early Caenorhabditis elegans embryos in mitosis.  Methods Cell Biol. 79: 101-19. Abstract Article Nicastro, D., Schwartz, C., Pierson, J., Gaudette, R., Porter, M.E. and McIntosh, J.R. (2006) The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313: 944-8. Abstract Article