Recent technological advancements in the 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.
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.
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.
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.