Electron microscopic visualization of collagen aggregates without chemical staining.

Lateral, uni-directional aggregates of collagen, segment-long-spacing crystallites (SLS), were made by dialyzing collagen solutions against acetic acid containing ATP, and were examined by transmission electron microscopes without electron staining. There detected were at least 20 dark (electron dense) cross-striations within SLS. The banding pattern was compared with the biochemical and biophysical properties of the amino acid side chains along the collagen molecule. The banding pattern of unstained SLS was mostly correlated with the intramolecular distribution of basic amino acid residues. Since basic amino acids have relatively large side chains, the distribution of mass (molecular weight of amino acids) along the molecule (local density profile) partially explained the cross-striation. Addition of fractions of molecular weight of ATP to basic residues made the local density profile much better correlate to the banding pattern. We conclude that the electron microscopic banding pattern of unstained SLS was generated by the local amount of material or local density profile along the collagen aggregates, which was enhanced by ATP bound to positively charged basic residues. The concept that the electron density correlates with the amount of material is fundamental in electron microscopy, and it has been proven with biological materials in this paper.