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3D Tomography and High-Resolution TEM

Photo of a FEI/Titan Krios 300kV TEM, which enables higher resolution for 2- and 3-D imaging of room-temperature or cryo specimens less than or equal to 1 micron.
FEI/Titan Krios 300kV TEM
Enables higher resolution for 2- and 3D imaging of room-temperature or cryo specimens less than or equal to 1 micron. Credit: NIAID

The EM unit has an FEI 120 kV Tecnai Spirit Biotwin and a FEI 300 kV Titan Krios TEM for 2D and cryo-tomographic capabilities to provide maximum ultrastructural 3D resolution to visualize viruses, bacteria, macromolecular complexes, and subcellular components for the study of host-pathogen interactions. This improved ability offers significant structural information, improving our ability to relate structure to function. The 300 kV microscope with automated stage, tomography package, and energy filtering systems improve resolution in all axes and give us the ability to bridge the imaging gap between confocal microscopy and electron microscopy and to expand toward molecular imaging.

3D Tomography

Tomographic rendering of Chlamydia trachomatis within an inclusion vacuole of a HeLa cell.
A three-dimensional tomographic reconstruction of a tick-borne flavivirus replication complex within the endoplasmic reticulum of a persistently infected tick cell.
Ortho slices and rendering of polio virus replication complexes in a HeLa cell.

Plunge Freezing

The Vitrobot allows rapid freezing of whole viruses, bacteria, and macromolecular complexes under climate-controlled conditions in liquid ethane cooled to liquid nitrogen temperatures.

Small aliquots of specimens in suspension are applied to holey grids, blotted with filter paper, and rapidly frozen to suspend within support film holes, where they are embedded in vitreous (non-crystalline) ice. Specimens can then be imaged under cryo-conditions for either 2D or 3D analysis.

After freezing specimens in suspension, regions of interest are determined under low-dose conditions to avoid radiation damage. Ideal imaging takes place in areas with very thin ice, usually where specimens are well-spread.

Three microscopic images of cryo-preserved Borrelia spp, Cryo-preserved mycobacteria spp, and cryo-preserved Chlamydiae spp.
Left to right: Cryo-preserved Borrelia spp.; Cryo-preserved Mycobacteria spp.; Cryo-preserved Chlamydiae spp. Credit: NIAID

Last Updated August 16, 2013

Last Reviewed August 16, 2013