Simple coherent anti-Stokes Raman spectroscopy system for minimally-invasive 3-D microscopy of lipid rafts in migratory cells

来自几个实验室的最新数据表明，细胞膜中的脂筏作为平台，允许正确的空间聚类和蛋白质的相互作用，这些蛋白质对于驱动定向细胞迁移至关重要。在其他功能中，脂筏结构与细胞内信号蛋白连接到膜上的特定位点，从而使生物活化成为可能。迄今为止，大多数脂筏的存在和作用的证据来自间接方法，如细胞膜的去污剂提取。由于荧光标记的困难，筏已被证明难以在活细胞中可视化。因此，寻求一种非侵入性的策略来研究迁移活细胞中的脂质结构。我们建议弥合这一技术和信息的差距，开发一个专用的仪器微创可视化的脂筏原位，使用相干反斯托克斯拉曼散射（汽车）显微镜。汽车显微镜是一种成熟的技术，它使用两个激光源以光谱方式确定样品的化学成分。在汽车成像中，两个光源在样品中相互作用以产生用于创建对比度图像的信号。信号可以从任何样品中产生，只要激光器可以提供激发分子振动所需的波长。此外，在汽车成像中不需要化学荧光标记，这减少了样品扰动。因此，汽车显微镜非常适合原位观察脂筏结构。目前汽车成像所采用的技术是昂贵的，高分辨率的，并且难以操作。通过开发一个简化和低成本的工作站，为传统和多重汽车显微镜使用光子技术的最新发展，我们将进行光谱成像的脂筏和进一步的结构，而不影响细胞活力。

Recent data from several laboratories has shown that lipid rafts in the cell membrane act as platforms to allow the correct spatial clustering and interaction of proteins essential for driving directed cell migration. Among other functions, lipid raft structures are linked with intracellular signalling proteins to specific sites at the membrane, thus enabling biological activation. To date, most of the evidence for the existence and role of lipid rafts has come from indirect methods such as detergent extraction of the plasma membrane. Rafts have proven difficult to visualise in living cells due to the difficulties associated with fluorescent labelling. A non-invasive strategy is therefore sought to investigate lipid structures in migratory living cells. We propose to bridge this technology and information gap by developing a dedicated instrument for minimally invasive visualisation of lipid rafts in situ, using coherent anti-Stokes Raman scattering (CARS) microscopy. CARS microscopy is an established technique that uses two laser sources to spectroscopically determine the chemical composition of a sample. In CARS imaging, the two light sources interact in a sample to generate a signal that is used to create a contrast image. A signal can be generated from any sample provided that the lasers can deliver the wavelengths necessary to excite the molecular vibration. Also, no chemical fluorescent labelling is required in CARS imaging, which reduces the sample perturbation. Hence, CARS microscopy is ideally suited to visualising lipid raft structures in situ. The current technology employed for CARS imaging is expensive, high-maintainance and is difficult to operate. By developing a simplified and low-cost workstation for both conventional and multiplex CARS microscopy using recent developments in photonics technology, we will perform spectroscopic imaging of lipid rafts and further structures without compromising cell viability.