Deuterium-Labeled Raman Microspectroscopic Study of Living Yeast Cells

• Main Authors: Chiang, Fu-Min, 蔣富珉
• Other Authors: Dr. Shigeto, Shinsuke
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/18568890619162694780

碩士 === 國立交通大學 === 應用化學系碩博士班 === 102 === Raman microspectroscopy provides powerful means to investigate cellular metabolism in living cells. When coupled with stable isotope labeling, it becomes even more powerful owing to the additional ability to trace intracellular metabolic dynamics. Recently we have demonstrated this novel approach using 13C-labeled glucose. In this study, we extend our work to deuterium (2H) labeling, which exhibits the largest isotope substitution effect because of a twofold difference in atomic mass. We measured a series of space-resolved Raman spectra from lipid droplets and the cytoplasmic (protein-rich) and cell wall (polysaccharide-rich) regions in living yeast cells at different incubation times in medium containing perdeuterated glucose as the primary carbon source. Upon 2H-isotope substitution, the C-H stretch band shifts from 3000–2800 to 2150 cm−1. Unlike other Raman bands in the congested fingerprint region (800–1800 cm−1), the C-D stretch band is easily discernible because it appears in the silent region, thereby facilitating the tracing of the time-dependent changes without ambiguity. In addition to the rise of the C-D stretch band, the ring breathing mode of the phenylalanine residues in proteins at 1003 cm−1 is found to downshift to two different wavenumbers, i.e., 961 and 975 cm−1. In order to explain this isotope substitution effect, we compare the experimental results with theoretical calculation results of the phenylalanine molecule whose ring hydrogen(s) are substituted with deuterium. We also present and discuss single-cell Raman imaging results.