Multiphoton microscopy of lipid-protein dynamics in living cells using correlative Coherent Antistokes Raman Scattering and Two-Photon Fluorescence

本研究的目的是开发一种新一代光学显微镜，能够分辨活细胞中脂滴(LD)及其相关蛋白的形成、运输和分解的动力学。学习障碍的发展在肥胖和糖尿病方面发挥着重要作用，这对现代发达国家的卫生预算造成了很大的负担。荧光显微镜，使用用染料标记的抗体或蛋白质与荧光标记的融合，提供了一种高度敏感和特定的方法来显示细胞中的蛋白质。可用于脂质光学显微镜的荧光标记是可用的，但存在快速光漂白的问题，即用光激发后荧光强度的不可逆降解。此外，由于添加荧光脂质探针和脂质染色过程而引起的细胞修饰提出了主要的问题，即所观察到的行为是真实的还是人为的。因此，用荧光显微镜很难研究激光二极管的发展和击穿动力学，而且往往不可能进行准确的定量。本项目中开发的显微镜的关键思想是通过脂类化学键的振动(拉曼散射)散射光来获得脂类的图像对比度。通过这种方式，细胞中存在的脂质被可视化，而不需要标记。当使用两个短激光脉冲激发振动并产生相干的反斯托克斯拉曼散射(CARS)时，可以增强拉曼散射。CARS非线性地依赖于激发光的强度，因此只有在激发光子集中的小焦域内才能获得足够的CARS产生强度。这导致了非常高的三维空间分辨率，并且可以用这种方法检测亚微米尺寸的脂滴。该显微镜将被设计成用CARS观察LD内部的脂质，并同时可视化与LD外部相关的标记蛋白的荧光，这样就可以在活细胞中确定LD发育的完整画面。我们已经确定了一些对LDS有影响的候选蛋白质。这台显微镜将使我们能够以一种以前不可能的方式来研究这些蛋白质和LDS相互作用背后的机制。人类细胞中负责LD动态平衡的蛋白质的识别将在设计控制细胞脂质含量的治疗策略中发挥关键作用。因此，该项目的较长期目标是开发一种高含量筛选版本的显微镜，以发现LDS中涉及的新靶点。除了直接参与该项目的研究人员外，卡迪夫大学的其他几位科学家还对与动脉粥样硬化、肺细胞产生表面活性物质防止肺塌陷、乳房发育和相关乳汁生产等相关的细胞脂感兴趣。这些研究人员将极大地受益于内部提供的专门用于脂质生物学的CARS/荧光显微镜，包括高含量筛选版本。这项工作的结果将发表在国际期刊上，这样来自世界各地物理和生物学科的研究人员将从这些结果中受益。这种显微镜技术的使用将在医学应用、药物发现和改善与脂质相关的健康问题的诊断和治疗方面具有重要意义。此外，本研究还为汽车显微镜的广泛应用提供了一种经济的设计方案。由于没有商用的汽车显微镜，我们预计显微镜制造商会对其开发感兴趣。此外，激光制造公司也将对实现用于汽车/多光子显微镜的激光源感兴趣。事实上，有一家这样的公司是这个项目的合作者。

The purpose of this research is to develop a new generation optical microscope able to resolve the dynamics of formation, trafficking and breakdown of lipid droplets (LD) and associated proteins in living cells. LD development plays a major role in obesity and diabetes which pose a substantial burden on modern developed countries health budgets. Fluorescence microscopy, using antibodies labelled with dyes or fusion of proteins with fluorescent tags has provided a highly sensitive and specific method of visualizing proteins in cells. Fluorescent markers for optical microscopy of lipids are available, however suffer from rapid photobleaching, that is an irreversible degradation of the fluorescence intensity after excitation with light. Moreover, the cellular modifications arising from the addition of fluorescent lipid probes and lipid staining processes raise major questions if the observed behaviour is real or artefactual. As a result the dynamics of LD development and breakdown has been very difficult to study with fluorescence microscopy, and accurate quantification often impossible. The key idea of the microscope being developed in this project is to obtain the image contrast from lipids via the scattering of light by the vibrations in the lipid chemical bonds (Raman scattering). In this way, the lipids present in cells are visualised without the need of labelling. Raman scattering can be enhanced when using two short laser pulses to excite the vibrations and generate Coherent Antistokes Raman Scattering (CARS). CARS depends nonlinearly on the exciting light intensity, so that sufficient intensities for CARS generation are achieved only in the small focal volume where the exciting photons are concentrated. This results in a very high three-dimensional spatial resolution and lipid droplets of submicron size can be examined with this method. The microscope will be designed to observe lipids on the inside of LDs with CARS, and simultaneously visualise the fluorescence from tagged-proteins associated to the outside of the LD, so that the complete picture of LD development can be determined in living cells. We have identified a number of candidate proteins that have an effect on LDs. This microscope will allow us to investigate the mechanisms behind the interactions of these proteins and LDs in a way that was previously not possible. The identification of the proteins responsible for LD homeostasis in human cells will play a key role in designing therapeutic strategies to control the cellular lipid content. Thus a longer term goal of the project is the development of a high-content screening version of the microscope to discover new targets involved in LDs. Besides the researchers directly involved in this project, several other scientists at Cardiff University are interested in cellular lipids in relation to e.g. atherosclerosis, pulmonary cells producing surfactants preventing lung collapse, breast development and associated milk production. These researchers would greatly benefit from the availability in-house of a CARS/fluorescence microscope dedicated to lipid biology, including a high-content screening version. Results of this work will be published in international journals, so that researchers from both physics and biological disciplines worldwide will benefit from these outcomes. The usage of this microscopy technique will be of relevance in medical applications, for drug discovery and to improve the diagnostic and treatment of lipid-related health problems. Additionally, the proposed research contains the realization of an economic design of the CARS microscope for its possible widespread application. With no CARS microscope commercially available, we expect microscope manufacturers to be interested in its exploitation. Also laser manufacturing companies will be interested in the realisation of laser sources optimised for CARS/multiphoton microscopy. In fact one such company is a collaborator on this project.