Objective: Small angle X-ray scattering (SAXS) analysis is a sensitive way of determining the ultrastructure of collagen in tissues. Little is known about how parameters measured by SAXS are affected by preservatives commonly used to prevent autolysis. We determined the effects of formalin, glutaraldehyde, Triton X and saline on measurements of fibril diameter, fibril diameter distribution, and D-spacing of corneal collagen using SAXS analysis.
Results: Compared to sections of sheep and cats' corneas stored frozen as controls, those preserved in 5% glutaraldehyde and 10% formalin had significantly larger mean collagen fibril diameters, increased fibril diameter distribution and decreased D-spacing. Sections of corneas preserved in Triton X had significantly increased collagen fibril diameters and decreased fibril diameter distribution. Those preserved in 0.9% saline had significantly increased mean collagen fibril diameters and decreased diameter distributions. Subjectively, the corneas preserved in 5% glutaraldehyde and 10% formalin maintained their transparency but those in Triton X and 0.9% saline became opaque. Subjective morphological assessment of transmission electron microscope images of corneas supported the SAXS data. Workers using SAXS analysis to characterize collagen should be alerted to changes that can be introduced by common preservatives in which their samples may have been stored.
Keywords: Collagen; Cornea; Formalin; Glutaraldehyde; SAXS; TritonX.