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Scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) have been carried out on hydrated fibrous assemblies of collagen I and pN-collagen I. STM of calf-skin collagen I that had been allowed to assemble on a graphite substrate showed t h e predicted 8 nm diameter microfibrils, arrays of which showed a 67 nm repeat. A detailed contrast variation along the microfilbrils was observed, corresponding to the established stain banding observed by transmission electron microscopy (TEM) and associated with hydrophilic, charged amino acid side chains along the staggered-molecule microfibril. This indicates that adsorbed water is an important mediator of STM imaging in insulators such as proteins. AFM images were obtained of D-periodic assemblies of pN-collagen deposited on carbon-coated copper TEM grids. The use of carbon-coated TEM grids as an SPM substrate allows experiments to be performed on the same samples, and correlation made between TEM and SPM images.

The egg case of the dogfish, Scyliorhinus canicula is a composite material largely constructed from collagen fibrils. It is formed as a drawn extrusion from transverse rows of spinnerets within the lining of the nidamental gland. In the L 2 layer, which forms over 90% of the thickness of the marginal rib, each spinneret extrudes a flattened ribbon which runs the length of the rib and measures approximately 150 x 8.5 μm in cross section. The structure of these ribbons and the orientation of collagen molecules and fibrils within them has been investigated in a correlative study using: low angle x-ray diffraction; bright field microscopy of peeled preparations; transmission (TEM) and scanning (SEM) electron microscopy; confocal and quantitative polarizing microscopy. The way in which the molecular orientations are defined within the spinneret has been followed by SEM of fixed material from actively secreting nidamental glands. The extruded ribbon showed a predominantly biaxial fibril orientation in low angle x-ray diffraction patterns recorded with the beam passing horizontally through the marginal rib. This x-ray pattern is derived from a remarkably regular parabolic arrangement of fibrils superficially resembling that seen in biological twisted nematic liquid crystals. However, evidence is presented here that the arrangement in the marginal rib is novel, apparently arising from authentically curved fibres showing a splayed- or bent- rather than twisted-nematic construction. Evidence is also presented that the spinnerets are able to control molecular orientations in a nematic liquid crystal to produce this and the other arrangements seen in the egg case.

Vertebrate mineralized tissues are composite materials formed by the organized growth of carbonated apatite crystals within a matrix of collagen fibres. Calcified collagen from turkey tendon was investigated using scanning tunnelling microscopy (STM) and atomic force microscopy (AFM). Samples were treated with hydrogen peroxide to enhance the mineralized phase by removing part of the collagen matrix and the results compared with the untreated material. Plate-like crystalline entities with dimensions 35 nm × 5-8 nm by ~ 1.5 nm were seen. These dimensions are consistent with previous reports using transmission electron microscopy (TEM) of calcified tendon and topographic imaging of tendon crystals. The resolution of the images obtained using STM is better than the previously reported pictures obtained using TEM or scanning electron microscopy (SEM). The value of 35 nm is the same as the gap region in the structure of the collagen fibrils. Stacking of plates and plateaggregates are a dominant feature in the scanning images. These results support the concept of organized intra-fibril mineral crystals within the organic collagen matrix. Electron diffraction and X-ray diffraction were undertaken on the samples and the patterns recorded match those previously reported for carbonated apatite.

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