Electron Microscope: Transmission, CRYO 2005

EMSL's T-12 cryo-TEM provides a unique capability for morphological studies by advanced imaging methods using cryostage and electron tomography.

Projections from a tilt series are collected and reconstructed into a volume using EMSL's T-12 cryo-TEM.

Whole mount of frozen-hydrated bacterium Shewanella oneidensis MR-1 as seen in a cryostage, showing exceptionally well-pronounced membranes and periplasmic space.

Ultracryo-microtomy is used at EMSL for obtaining frozen-hydrated sections for immuno-cytochemistry studies and cryo tomography.

In 2006 EMSL brought online an FEI Tecnai T-12 cryo-transmission electron microscope (TEM) that complements the user facility's state-of-the-art electron microscopy suite and JEOL 2010 analytical high-resolution TEM. The cutting-edge capabilities of the T-12 cryo-TEM include: (1) a high-tilt stage that allows image tilt series to be obtained for three-dimensional reconstruction using electron tomography, and (2) a cryostage for observing samples in a frozen-hydrated state. The T-12 cryo-TEM is primarily devoted to biological samples involving morphological and immuno-cyto chemistry studies, but it also supports imaging of samples such as soft materials and polymers.

Electron Tomography

EMSL's T-12 cryo-TEM is customized to accommodate its users' demands within the fast-growing field of biological imaging at the nano-structural level. Coupled with electron tomography capabilities, three-dimensional reconstruction of an object that was acquired from a series of two-dimensional projections using TEM can be achieved. The object is successively rotated along one or two axes (± 70 degrees) and digitally imaged; the images are then reconstructed into the object's three-dimensional structure. The computed back-projection can be viewed as a slice-by-slice virtual sectioning through the reconstructed volume. A dramatic increase in resolution is achieved when compared with traditionally prepared ultra-thin plastic TEM sections.

Cryostage

The observation of frozen-hydrated samples is playing an increasingly important role in structural biology imaging, despite the great difficulty and limitations typically encountered. However, through EMSL's T-12 cryo-TEM and associated sample preparation suite, including Vitrobot and ultracryo-microtome, rapid freezing of cells and tissues provides outstanding structure preservation and good time resolution of dynamic cellular processes in situ. In addition, the fragile chemical balance of water-soluble protein complexes that is compromised by conventional organic solvent-based TEM methods can now be better preserved, resulting in detailed insight into the structural basis and ultimately the function of cellular processes.

Advanced Methods for Sample Preparation

EMSL's TEM laboratory is equipped with a suite of instruments for traditional and modern sample preparation. The instrumentation includes a Leica EM Pact high-pressure freezer, an automatic freeze substitution unit, ultramicrotomes with a cryo chamber for frozen-hydrated sections preparation, and a Vitrobot for vitrification of suspension for cryostage observation.

Specifications

• High-contrast, high-resolution imaging for 20 kV to 120 kV operation
• Optimized for two- and three-dimensional imaging of cells, cell components, and soft materials
• ± 70 degrees fully automated tilt series acquisition for electron tomography
• Liquid nitrogen temperature observation of frozen-hydrated samples
• Low-dose observation and imaging.

User Project Areas

• High-resolution imaging and analyses of bacterial-mineral interactions
• Morphological studies using electron tomography
• Immunogold labeling of ultracryo or plastic sections for localization of immune response
• Cryostage observation of dynamic cellular processes

Related Publications

1. Ligand field effects on the multiplet structure of the U4f XPS of UO2.
2. A New Analysis of X-Ray Adsorption Branching Ratios: Use of Russell-Saunders Coupling .
3. In Vitro Cell Culture Infectivity Assay for Human Noroviruses.
4. The effect of U(VI) bioreduction kinetics on subsequent reoxidation of biogenic U(IV).
5. c-Type Cytochrome-Dependent Formation of U(IV) Nanoparticles by Shewanella oneidensis .