Infrared Microspectroscopy and Laser Raman Microprobe in the Diagnosis of Cystinosis

Abstract

Infrared microspectroscopy and the laser Raman microprobe method have been employed for the identification of cystine crystals in human tissue specimens. Scanning electron microscopy was also employed to study the morphologic changes associated with the formation of these crystals in tissue. For the Raman microprobe experiments, unstained tissue specimens sectioned at 6 to 10-μm were mounted on an ordinary microscope glass slide and excited with the 514.5-nm line from an argonion laser. Cystine crystals in liver and spleen specimens were easily identified by the enhancement of Raman lines at 501 and 2916–2967 cm−1 arising from the disulfide (S-S) linkage and the C-H stretch motion, respectively. The infrared spectra, on the other hand, displayed absorptions at 1338, 1411, and 1621–1656 cm−1 assigned to the cystine C–C, –COO−, and C=O group stretching frequencies, respectively. In contrast to previous macro-analytical applications of Raman scattering and infrared spectroscopy, the micro-analytical experimental approach developed here allows for morphologic and analytical determinations to be conducted on the same tissue specimen. In addition, because of the nondestructive nature of Raman and infrared microspectroscopy, the combination of these two techniques provides a rapid, accurate, and sensitive analysis to improve the diagnosis of these materials in tissue sections.