(ii) New dual beam technology for automated, site-specific 3D imaging of cells and tissues
see Heymann et al (2006)

The conventional method for imaging the interior of cells and tissues by transmission electron microscopy is to prepare thin sections, which can then be imaged in projection. In a collaborative project with FEI Inc., we are developing a new approach that allows the sectioning of cells and tissues to be carried out electronically inside the specimen chamber using a strong ion beam than can remove material from the surface of biological material in increments as small as ~50 nm. The first step in the imaging process is to excavate the selected region of the specimen with the focused ion beam. The exposed surface is then imaged at higher magnifications with a scanning beam and can produce images at resolutions about ten times better than with conventional optical microscopy (panel at lower left). By iterating ion beam milling with surface imaging, a 3D reconstruction of the object can be obtained. The dual beam imaging methods we are developing complement the electron tomographic approaches we routinely use in the lab. The goal of the cryo electron tomography is to achieve the highest possible resolutions from very small volumes (typically 1 micron wide and ~ 2000 Å thick), good enough to interpret in the context of the molecular structures of individual proteins. In contrast, the goal of dual beam imaging is the rapid, and lower resolution imaging of cells and tissues (typically of volumes 20 um x 20 um x 20 um) in size and to achieve resolutions that are about 5-10 times higher than confocal microscopy.

(iii) Use of electron dense labels for 3D molecular localization studies using tomography
see Zhang et al (2005).

	3D localization of ferritin in degenerating mouse neurons.

It is highly desirable to develop methods for directly localizing specific molecules in 3D in electron tomograms along the same lines that is possible for 3D visualization of GFP-labeled proteins using confocal microscopy. Visualization of suitably tagged molecules at the ~10 nm resolutions that can currently be achieved by electron tomography could revolutionize our understanding of the dynamic protein-protein interactions that occur in cells. We have explored the potential of exploiting ferritin for such studies, because of its electron dense core of ~ 3000 iron atoms.

This exploration has led unexpectedly to an interest in understanding the structural basis of degeneration in mouse neurons, as demonstrated in the paper cited above. In this work, we showed that the 3D location of ferritin molecules in tissue could be directly determined in 3D. We demonstrate that instead of being present in the interior of the axon, as had been concluded from previous studies using optical microscopy, ferritin is located in oligodendrocyte cells that happen to invade the milieu of the degenerating axons. This ten-fold increase in resolution with electron tomography has thus led to a completely different mechanism for neuronal degeneration than one might have concluded based on the optical microscopic studies alone.