An FT-IR microscopic investigation of the effects of tissue preservation on bone

Fourier transform infrared microscopy is a powerful tool for the characterization of mineral and protein in histologic sections of bone. This study was concerned with determining whether techniques used to preserve these tissues and to prepare them for sectioning had an effect on spectral properties. The nu 1, nu 3 phosphate bands in the 900-1200 cm-1 spectral region were used to evaluate the structure of the apatitic mineral in fresh-frozen, ethanol-fixed, and formalin-fixed 35-day-old rat femurs; fresh-frozen and formalin-fixed 20-day-old fetal rat femurs; ground 35-day-old rat diaphyseal bone samples; and formalin-fixed, methacrylate-embedded ground diaphyseal bone. The crystallinity (crystal size and perfection) of the bone apatite was assessed by a curve-fitting analysis of the nu 1, nu 3 phosphate bands. Results indicate that ethanol or formalin fixation of the 35-day-old intact rat femur, and formalin fixation and embedding of the ground rat bone do not significantly alter the crystallinity of the apatite. However, formalin fixation of the fetal rat bone did alter the structure of the apatite mineral phase. In addition, evaluation of protein secondary structure in the 35-day-old rat femur from the Amide I and Amide II vibrations near 1650 and 1550 cm-1, respectively, revealed that protein conformation was altered by ethanol fixation.