利用顯微拉微光譜和拉曼成像法研究大腸桿菌生物膜和利用同位素標記法研究活體裂殖酵母菌細胞代謝動力學

Abstract

Biological processes have intricate designs by nature. The main purpose of this work is to decode some of these designs. Many areas in biology including studies on microbial communities and cellular biochemistry still largely remain unexplored due primarily to the lack of appropriate tools. In this thesis, we present the applications of Raman micro-spectroscopy and imaging to gain fundamental and otherwise unobtainable biological information on complex structured communities of bacteria known as biofilms and on the metabolic dynamics in single yeast cells.

To demonstrate the power of Raman spectroscopy for complex biological systems, model Escherichia coli biofilms were studied. A variety of biomolecules have been shown to play a unique role as signals and/or regulators in biofilm formation. By using Raman imaging,we investigated model Escherichia coli biofilms and detected high levels of he amino acid leucine accumulation during the early stage of the E. coli biofilm formation, which may have resulted from physiological environment-specific metabolic adaptation. Our results demonstrates that our label-free Raman imaging method provides a useful platform for directly identifying still unknown natural products produced in biofilms as well as for visualizing heterogeneous distributions of biofilm constituents in situ.

To elucidate the dynamics of intracellular proteins and lipids at the single cell level, the Raman method was coupled with a very powerful strategy, namely, stable isotope labelling. Here, we present in vivo time lapse Raman imaging, coupled with stable-isotope (13C) labelling, of single living Schizosaccharomyces pombe cells. Lipid droplets have been hypothesized to be intimately associated with intracellular proteins. However, there is little direct evidence for both spatiotemporal and functional relations between lipid droplets and proteins provided by molecular-level studies on intact cells. Using characteristic Raman bands of proteins and lipids, the process by which 13C-glucose in the medium was assimilated into those intracellular components was dynamically visualized. Our results show that the proteins newly synthesized from incorporated 13C-substrate are localized specifically to lipid droplets as the lipid concentration within the cell increases. We demonstrate that the present method offers a unique platform for proteome visualization without the need for tagging individual proteins with fluorescent probes.

Finally, in chapter V we conclude by summarizing what has been achieved during this thesis work and also present a possible new direction of study in the future with our method.

Biological processes have intricate designs by nature. The main purpose of this work is to decode some of these designs. Many areas in biology including studies on microbial communities and cellular biochemistry still largely remain unexplored due primarily to the lack of appropriate tools. In this thesis, we present the applications of Raman micro-spectroscopy and imaging to gain fundamental and otherwise unobtainable biological information on complex structured communities of bacteria known as biofilms and on the metabolic dynamics in single yeast cells.