Reference sample method reduces the error caused by variable cryosection thickness in Fourier transform infrared imaging.

Fourier transform infrared imaging (FT-IRI) is a novel technique for characterization of the biochemical composition of biological tissues, e.g., articular cartilage. The use of cryosections is preferred in FT-IRI. Unfortunately, significant variation in section thickness often impairs the suitability of cryosections for quantitative FT-IRI analysis. The present study introduces an inexpensive reference sample method for quantitative analysis. In this technique, specimen absorption is normalized with that of nitrocellulose membrane embedded and cryosectioned with the sample. Mean variation of the infrared absorption in cartilage specimens was 11.5%, 12.1%, and 20.6% for 5 microm, 10 microm, and 14 microm thick sections, respectively, without normalization. Normalization reduced the variation to 5.2%, 4.0%, and 4.6% for the same sections, respectively. The normalization method enables usage of cryosections for quantitative work and significantly reduces the cost and time needed for FT-IRI analysis.