Applications of coherent anti-Stokes Raman scattering microscopy to the research of lipid biology
| Autor: | Cheng-Hao Chien, 錢正浩 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 Coherent anti-Stokes Raman scattering (CARS) microscopy is an optical imaging technique based on Raman vibrational modes of molecules. The advantages of CARS microscopy include label-free, non-invasive, and optical sectioning ability. It is a powerful imaging tool for biomolecules, especially lipids, in living cells and animals. In this thesis, we will present the construction of CARS microscopy and its application to the research of lipid biology in Drosophila, atherosclerosis, and Caenorhabditis elegans (C. elegans). In the study of Drosophila, we used CARS and two-photon excitation fluorescence (TPEF) microscopy to acquire sectioning images of a living larva for the first time. We monitored fat body remodeling process at subcellular level and found an increase in autofluorescence globules associated with a degradation of LDs in each fat cell, which indicated the recruitment of proteins in fat cells as well as the consumption of lipid. Moreover, we observed the metabolism of hepatocyte-like cell oenocytes and the fat body as the imbalance of two lipid regulatory proteins, Bmm and Lsd-2. This in vivo observation of oenocytes and fat body provided direct information about lipid homeostasis in Drosophila. In the study of atherosclerosis, we focused on the foam cell formation at the beginning of atherosclerosis, by monitoring lipid accumulation and hydrolysis in living macrophages. We further analyzed kinetic properties of the inhibitor diethylumbelliferyl phosphate (DEUP) and its inhibition efficiency. This approach can further be applied to the study of lipid metabolism in other cellular systems, and the high throughput screening of therapeutic agents for lipid metabolic diseases. In the study of C. elegans, we characterized lipid distribution at each larval stage and found lipid reallocation during development of L4 larva to adult, which implies that the consumption of lipid is for developing gonad. We further observed lipid droplet patterns in fat mutants, and found yolk lipoprotein accumulation without labeling for the first time. These results showed that in addition to lipid, CARS microscopy can also provide in vivo label-free imaging of lipoproteins in C. elegans. Our work demonstrated that by using CARS microscopy, we could investigate lipid distribution, lipid content, and lipid dynamics in living organisms, which can offer more information for the research of lipid biology. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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