Tissue acquisition and storage associated oxidation considerations for FTIR microspectroscopic imaging of polyunsaturated fatty acids

The dietary intake of polyunsaturated fatty acids (PUFAs), e.g., docosahexaenoic acid, eicosapentaenoic acid and arachidonic acid, is reflected in the biochemical makeup of tissues. In the course of analyzing mouse retina and brain tissue with Fourier Transform infrared (FTIR) microspectroscopy, we discovered that special considerations are required to minimize loss of PUFA. Traditionally, imaging of the 3012 cm−1 band (olefinic C–H stretch) has been carried out on cryosectioned tissues that have been mounted on appropriate substrates and stored in dark, dry, room temperature conditions prior to data collection. This storage condition is inadequate, due to surprisingly rapid oxidation of the CC–H functional group in PUFA and to a lesser extent, of lipid carbonyl, CO. Olefinic degradation is illustrated by the decrease in intensity of the 3012 cm−1 peak over time in the rod cell outer segment (ROS) in retina, a region that contains highly concentrated PUFA. Time-lapse experiments on samples stored under traditional conditions illustrate that the storage lifetime is limited to a few days. We have tested new protocols for sample preparation and storage that permit more accurate assessment of PUFA in biological tissues by FTIR analysis. This information will enable larger ongoing research efforts into the analysis of PUFA in biological tissue samples with FTIR microspectroscopy in healthy and diseased animal models.