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

使用相关相干反斯托克斯拉曼散射和双光子荧光对活细胞中的脂质蛋白动力学进行多光子显微镜观察

• 批准号: BB/H006575/1
• 负责人: Paola Borri
• 金额: $ 58.74万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH006575%2F1
• 关键词: Multiphoton; microscopy; lipid; protein; dynamics

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.

本研究的目的是开发一种新一代光学显微镜，能够解决活细胞中脂滴（LD）和相关蛋白的形成，运输和分解动力学。低收入国家的发展在肥胖和糖尿病中起着重要作用，这对现代发达国家的卫生预算造成了沉重负担。荧光显微镜，使用染料标记的抗体或蛋白质与荧光标签的融合，提供了一种高度敏感和特异性的方法来观察细胞中的蛋白质。用于脂质光学显微镜的荧光标记物是可用的，但是遭受快速光漂白，即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引起的细胞修饰提出了主要问题，如果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LD的发展和分解动力学，而且往往无法精确定量。在这个项目中开发的显微镜的关键思想是通过脂质化学键振动的光散射（拉曼散射）来获得脂质的图像对比度。这样，细胞中的脂质就可以可视化，而不需要标记。利用两个短激光脉冲激发振动并产生相干反斯托克斯拉曼散射（CARS）可以增强拉曼散射。CARS非线性地依赖于激发光强度，因此只有在激发光子集中的小焦体中才能产生足够的CARS强度。这导致了一个非常高的三维空间分辨率和亚微米大小的脂滴可以用这种方法进行检查。该显微镜将被设计用于用CARS观察LD内部的脂质，同时观察LD外部相关的标记蛋白的荧光，以便在活细胞中确定LD发展的完整图景。我们已经确定了一些对ld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研究这些蛋白质和ld相互作用背后的机制。鉴定人类细胞中负责LD稳态的蛋白质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，该项目的长期目标是开发高含量筛选版本的显微镜，以发现ld中涉及的新目标。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也对细胞脂质与动脉粥样硬化、肺细胞产生表面活性剂防止肺塌陷、乳房发育和相关的乳汁产生等方面的关系感兴趣。这些研究人员将极大地受益于内部专用于脂质生物学的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，以便世界各地的物理和生物学科的研究人员将从这些成果中受益。这种显微技术的使用将在医学应用中具有相关性，用于药物发现和改善与脂质相关的健康问题的诊断和治疗。此外，提出的研究包括实现汽车显微镜的经济设计，为其可能的广泛应用。由于目前市面上还没有car显微镜，我们希望显微镜制造商对其开发有兴趣。此外，激光制造公司将有兴趣实现优化的激光光源的CARS/多光子显微镜。事实上，其中一家公司就是这个项目的合作者。

项目成果

Quantitative coherent Raman scattering microscopy for bioimaging

用于生物成像的定量相干拉曼散射显微镜

• DOI: 10.1109/cleo/europe-eqec52157.2021.9542671
• 发表时间: 2021
• 作者: Borri P

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

• DOI: 10.1242/dev.129908
• 发表时间: 2016-06-15
• 期刊: Development (Cambridge, England)
• 作者: Bradley J;Pope I;Masia F;Sanusi R;Langbein W;Swann K;Borri P
• 通讯作者: Borri P

Optimisation of multimodal coherent anti-Stokes Raman scattering microscopy for the detection of isotope-labelled molecules

• DOI: 10.1117/12.2509280
• 发表时间: 2019-03
• 作者: Dale Boorman;I. Pope;W. Langbein;S. Hood;P. Borri;P. Watson

Quantification of the nonlinear susceptibility of the hydrogen and deuterium stretch vibration for biomolecules in coherent Raman micro-spectroscopy.

定量氢和氘拉伸振动对相干拉曼微光谱法中生物分子的敏感性。

• DOI: 10.1002/jrs.6164
• 发表时间: 2021-09
• 期刊: JOURNAL OF RAMAN SPECTROSCOPY
• 影响因子: 2.5
• 作者: Boorman, Dale;Pope, Iestyn;Masia, Francesco;Watson, Peter;Borri, Paola;Langbein, Wolfgang
• 通讯作者: Langbein, Wolfgang