Timothy J. O'Leary, Walter F. Engler, Kathleen M. Ventre
Proceedings Volume 7th Intl Conf on Fourier Transform Spectroscopy, (1989) https://doi.org/10.1117/12.969588
Infrared spectroscopy is a powerful technique by which to characterize the conformations of proteins, lipids, and nucleic acids (1). Previously we have demonstrated that infrared spectroscopy can be used to characterize the secondary structure of abnormal protein accumulation products, known as amyloid, which are often found in association with medullary carcinoma of the thyroid (2). The utility of the technique was highly limited by the fact that essentially the entire specimen had to consist of this abnormal protein for infrared spectroscopic analysis to be useful. The development of high quality microscopes capable of both light microscopic and infrared characterization of materials has enabled us to extend our earlier use of infrared spectroscopy to diseases and tissues in which the abnormal region of interest is only a few hundred square micrometers in area. Tissue for spectroscopic examination is mounted on microscope slides which have been prepared by acid washing, plating with gold or gold-palladium alloy (3) and coating with high molecular weight poly-L-lysine. Sections of tissue which have been previously embedded in paraffin are cut with a microtome at 4 to 5 micrometers thickness, floated onto a bath of distilled water, picked up on the microscope slide, and allowed to dry overnight. Paraffin is removed by soaking the slides in two changes of xylene, and then the sections are rehydrated by placing them in absolute alcohol, then in fifty percent alcohol, and finally in water. Sections may then be stained using standard histologic stains, such as hematoxylin and eosin, then once again dehydrated with alcohol. After drying, the sections are covered with an index-matching fluid, such as Fluorolube, which allows a relatively good visual microscopic examination of the tissue when the microscope is used in reflectance mode. High quality reflectance infrared spectra may be easily obtained when the tissue is prepared and mounted in this way (Figure 1). Alternatively, fresh or formalin-fixed tissues which have not been embedded in paraffin may be prepared for examination by freezing them in a cryostat, cutting five to ten micrometer thick sections, and mounting them directly on the polylysine coated gold-plated slides. These tissues may then be stained with hematoxylin and eosin or with Diff-Quik, then dehydrated with acetone, thus preserving cellular lipids. We have examined a number of cases of medullary carcinoma of the thyroid and obtained infrared spectra of the associated amyloid protein. Spectra were obtained using an IR-Plan microscope interfaced to a Bomem DA3 Fourier transform infrared spectrometer. A 32x objective was used, with a circular aperture which allowed acquisition of spectra from a region as small as 90 micrometers in diameter. A narrow-band 0.25 mm MCT detector was employed. A typical spectrum from amyloid found i11 a medullary carcinoma of the thyroid is found in Figure 1; features found in the 1630 to 1645 cm region of the Amide I band are indicative of β-sheet structure, which has previously been described in amyloid proteins (4). The amount of fl-sheet structure, as assessed visually in comparison with the rest of the Amide I band, varies markedly from region to region and case to case. The presence of this βsheet structure cannot be used to differentiate amyloid from other extracellular proteins. Figure 2 shows the spectrum of colloid from a thyroid follicle. This material, which is largely composed of thyroglobulin, also shows a significant amount of βstructure, as does the heart muscle examined following frozen section. In the case of the heart muscle, however, cellular lipid is also observed as methylene C-H stretching modes in the 2800-3100 cm region of the spectrum. The frozen section tissue preparation procedure leaves the cellular lipid in place, while the paraffin-embedding and removal procedure used for preparation of the first two specimens extracts cellular lipids as well, resulting in a much less prominent C-H stretching mode region.
