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3D structure of trimeric HIV-1 spike

3D structure of trimeric HIV-1 spike. See article by Liu et al (2008).

Press Release: Structure of trimeric HIV-1
1. NCI News

Architecture of simian immunodeficiency virus determined using cryo-electron tomography

Architecture of simian immunodeficiency virus determined using cryo-electron tomography. See article by Sougrat et al (2007).


Press Releases: HIV Molecular Entry Claw
1. NIH News
2. NIH Research Matters

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RESEARCH

Chemotaxis | HIV Imaging | Molecular Machines | Nanobiology

Nanobiology and new technologies for 3D imaging

We are working on a wide spectrum of technical advances aimed at sharpening the tools that will be essential to realize the promise of imaging cells and tissues at molecular resolution with electron-based imaging technologies. Some of these are in-house projects, and others involve formal collaborative partnerships with industry. Representative projects are listed below:

(i) Development of new specimen stages for high-throughput electron microscopy
see Lefman et al (2007)

Gatling gun
Presently available methods for loading specimens into transmission electron microscopes are usually time-consuming and require manual intervention. In collaboration with GATAN Inc., we have developed a novel cartridge based system, which allows for up to 100 specimens to be loaded into the microscope simultaneously (see view of specimen chamber on right) and provides for fast, automated specimen exchange. The use of this system should greatly increase the throughput of specimens for transmission electron microscopy and tomography. We are also in early stages of evaluation of other novel types of specimen stages such as cylindrically symmetric holders for full tilt tomography that we have already shown to be functional in prototypes constructed in our lab.
   

(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.

Cryo electron tomography

 

 

(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 neuron.
 

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.

 

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