Skip Navigation

Translational Research Tools and Services

Skip Content Marketing
  • Share this:
  • submit to facebook
  • Tweet it
  • submit to reddit
  • submit to StumbleUpon
  • submit to Google +

Cryo-Electron Microscopy

Recent technological advancements in the NIAID Rocky Mountain Laboratories Microscopy Unit have introduced advanced preparative technologies and techniques for cryo-immobilization (freezing) of biological specimens. The results show that improvements in retaining components often lost during routine processing can allow near native-state observation of specimens, increasing our ability to more clearly relate structures to function. Below are examples of our current developments in various cryo-techniques.

The Microscopy Unit has designed, developed, and deployed a cryo-thermal device for greatly accelerating freeze substitution processing of biomaterials for light and electron microscopy in a process called microwave-assisted freeze substitution.

High-Pressure Freezing

Four electron microscope images of high-pressure freezing
High-pressure freezing with a Leica EMPact high-pressure freezer and freeze fracturing with a Gatan Alto 2500 cryo-system on a Hitachi S-4500 scanning electron microscope revealed intracellular spatial information of HeLa cell and chlamydial interactions with improved structural integrity (Fig. a, b). Extraction of cytoplasmic components by partial fixation prior to plunge freezing exposed structures for improved depth of visualization within the bacterial inclusion (c). TEM of high pressure frozen and freeze-substituted chlamydia revealed spike-like structures consistent with secretion apparatus (d).

Freeze Fracture

Viewing intracellular details of pathogens and their relationships to membranous compartments by cryo-SEM after high-pressure freezing becomes difficult when fixed cytoplasm obscures the areas of interest. Combined techniques of chemical fixation and cryo-fracturing for viewing by cryo-SEM improved visualization of interactions between intracellular pathogens and their membrane-bound compartments in tissue culture cells.

Microscopic images of Coxiella burnetii infected Vero cells (a) and Francisella tularensis infected mouse macrophages (b) after chemical fixation, plunge freezing, fracturing, and viewing by cryo-SEM.
Coxiella burnetii infected Vero cells (a) and Francisella tularensis infected mouse macrophages (b) after chemical fixation, plunge freezing, fracturing, and viewing by cryo-SEM.

Cryo-Sputter Coating

The BAF 060 freeze fracture device allows dual target rotary shadowing of metals that prevents charge artifact without obscuring structural details. The BAF 060 has the unique ability to apply coatings at varied angles to produce a shadow effect under cryo-conditions, allowing direct viewing of bulk samples in near-native state, with a much increased capacity to resolve minute details for both direct viewing with a cryo-SEM stage or generation of replicas that can be viewed by transmission electron microscopy, as shown.

Images of Bal-tec BAF 060 freeze-fracture device carbon/platinum replica of high-pressure frozen and cryo-fractured yeast (a-e) and Bacillus subtilis (f-g) visualized by TEM.
Bal-tec BAF 060 freeze-fracture devicecarbon/platinum replica of high-pressure frozen and cryo-fractured yeast (a-e) and Bacillus subtilis (f-g) visualized by TEM.

Cryo-Ultramicrotomy

For antigens that are soluble or sensitive to chemicals during fixation steps, cryo-preservation becomes a useful alternative for immuno-labeling, using Leica UCT cryo-ultramicrotome.

As shown below, after cryo-sections are retrieved, fluorescent labeling can be accomplished on sections adhered to glass slides to test antigen preservation.

microscopic images of Cryo-sections of HeLa cells infected for 20 hours with Chlamydia trachomatis
Cryo-sections of HeLa cells infected for 20 hours with Chlamydia trachomatis. (Left) Light micrograph of frozen section cut at 300 nm. (Middle) Light micrograph of 300 nm thick section stained with anti-chlamydial and fluorescent antibodies. (Right) Transmission electron micrograph of 50 nm frozen-hydrated thin sections labeled with anti-chlamydial and 10 nm gold antibodies.

Last Updated August 16, 2013

Last Reviewed August 16, 2013