Abstract
Lipids have various crucial functions in biological systems, such as being the building blocks of membranes, providing as fuel molecules, and acting as signaling molecules(such as steroid hormones). However, the excess intake of lipids can be one major reason that causes several chronic diseases, such as atherosclerosis, cardiovascular disease, chronic liver or kidney diseases. Therefore, it is important to study and understand the lipid metabolism in the biological systems. For a long time, lipids have been regarded as “the invisible molecules” for in vivo observation because they are transparent, intrinsically non-fluorescent, and difficult to be tagged with fluorophores. Coherent Raman technique, such as coherent anti-Stokes Raman scattering (CARS) microscopy, provides a label-free way to specifically visualize biomolecules, particularly for detecting lipids, in living organisms at subcellular resolution, which makes it a powerful tool for the study of lipid metabolism and lipid biology. In this thesis, we will present the construction of CARS microscopy and the studies of lipid metabolism in the live cell system (macrophages) and in live animal model (C. elegans). The accumulation of lipid in macrophages is a key factor that promotes the formation of atherosclerotic lesion. In the study of live cell system (macrophages), we developed an automated quantitative analysis method for the real-time assessment of lipid content in living macrophages. This method can monitor the lipid accumulation and hydrolysis in the living cells in real time at single-cell level without any labeling. We showed that this method can also be applied to other type of cells such as lung cancer cells (CL1-0), which broadens the possible applications of this method. Finally, we demonstrated its potential for kinetic study of drugs and for high-throughput screening of lipid therapeutic agents. To verify the question – whether the composition of fatty acids affects the uptake of lipoprotein, we chose C. elegans as a model animal for the study because the yolk lipoprotein in C. elegans is homologous human low-density lipoprotein (LDL) and both of them (yolk lipoprotein and LDL) act the same function, as the major lipid carrier delivering lipid into cells. In the study of live animal model (C. elegans), we first demonstrated that the abnormal accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm−1) and lipid (~2845 cm−1) Raman bands. In addition, we developed image analysis protocols to quantitatively measure the abnormal accumulation of secreted yolk lipoprotein and the oocyte lipid content in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results reveal that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This thesis work not only demonstrates that CARS microscopy is a useful technique for the label-free examination of lipid metabolism in live biological systems but also elucidates that omega-6 PUFA is a key factor that strongly influences the uptake of lipoprotein in the biological systems. We believe that the extensive and intensive researches based on live cell/animal model will help us to understand the genetics as well as metabolic pathways in lipid biology, and will possibly find a way to tackle human lipid metabolic diseases in the future.