title: Cryo-TEM analysis of collagen fibrillar structure.
abstract: Fibrillar collagens are important structural proteins and are known to be closely associated with mineral in the case of mineralized tissues. However, the precise role of collagen in the mineralization process remains unclear, and the evaluation of structural differences in collagen from mineralized and nonmineralized tissues may be instructive in this regard. Here, we review the use of cryo-transmission electron microscopy to investigate the axial structure of collagen fibrils in tissue sections from both mineralizing and nonmineralizing tissues. By examining collagen fibrillar structure in an unstained frozen-hydrated state, it is possible to avoid artifacts normally associated with staining and dehydration that are required for conventional TEM. We describe both sample preparation and image analysis with emphasis on the particular challenges of using image averaging techniques, which can be used to overcome the low signal-to-noise ratio that is inherent in this technique. Detailed banding patterns can be obtained from averaged images, and these can be analyzed to obtain quantitative information on fibril periodicity and structure.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Quan
authors.forename: Eli D
authors.initials: ED
authors.lastname: Sone
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy; D003804:Dentin / Q000648:ultrastructure; D024022:Fibrillar Collagens / Q000648:ultrastructure*; D005629:Frozen Sections; D008297:Male; D051379:Mice; D046529:Microscopy, Electron, Transmission; D010513:Periodontal Ligament / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D012867:Skin / Q000648:ultrastructure; D013710:Tendons / Q000648:ultrastructure; D058989:Vitrification
chemical_list: D024022:Fibrillar Collagens
keywords: Collagen banding; Connective tissues; Cryo-transmission electron microscopy; D-spacing; Frozen-hydrated sections; Image averaging