Spatially Resolved Improved FT–IR Microspectroscopy of Deuterated Species in Tissue
David L. Wetzel, Steven M. LeVine
2002
Microscopy and Microanalysis
Stretching vibrations of CH, NH, and OH observable with FT-IR microspectroscopy have allowed a means of localizing compounds containing deuterium isotopes in plant and mammalian tissue. Relatively high detection limits heretofore have required thicker than usual sections or a sufficient quantity of deuterated compound applied or accumulated in the tissue to allow detection and reliable peak area determination. This paper is concerned with spectra obtained with a recently improved instrumental
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... pability that has reduced noise and increased detector sensitivity in the 2600-2000 cm -1 region of the spectrum. Previous Work: The migration of water in wheat kernels during the tempering process prior to flour milling was studied. Wheat kernels soaked in D 2 O were withdrawn at different time intervals, frozen, sectioned, placed between barium flouride windows, and immediately li ne mapped to trace the movement of the tempering heavy water in the presence of natural water already in the seed [1, 2] . Twenty micrometer thick sections gave measurable OD peak areas while other absorption bands were off scale. Metabolically incorporated CD and ND/OH deposits in the cerebella of adult rats that had 30-40% D 2 O in their drinking water for 5½ weeks were examined. A multitude of spectra from line mapping across layers of white matter, nuclear cell layer, and molecular cell layer showed the deposition of the CD and combined ND, OD bands for each of the three different layers. The hydrophobic white matter had the greatest amount of CD and hydrophilic nuclear cell layers had the greatest amount of the other deuterated forms. The potential for the study of brain metabolism was suggested by comparison of the peak area of CD with CH etc. to reveal uptake of the deuterium isotope from the ingested D 2 O [3]. The spot size analyzed with a synchrotron illuminated infrared microspectroscope was 12 Φm x 12 Φm. Additional spectra were collected using an infrared microscope with a conventional globar source from 36 Φm x 36 Φm spot areas. These latter data were used for statistical treatment. Preliminary Observations and Application of the Current Instrumental Capability: Intermediate to the work reported in the past with the wheat kernels and rat cerebella preliminary experiments with deuterated organic compounds and animal tissue with a conventional globar source and thin, 8 Φm, sections showed the need for lower detection limits. Several instrument modifications were made and further experimentations obtained spectra from neat deuterated organic compounds as well as the same compounds on a tissue substrate. Also, deuterated compounds that were applied topically or systemically were found subsequently by observation of the CD absorption band detected above baseline noise in tissue from the experimental animals. These data indicated the potential of the microspectroscopic technique with improved detection limits. The instrument in current use is a ContinuΦm ® equipped with a
doi:10.1017/s1431927602104144
fatcat:igfn2f77njgyfamyrggyvbbg5y