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

最近来自几个实验室的数据表明，细胞膜上的脂筏作为平台，允许正确的空间聚集和蛋白质之间的相互作用，这是驱动细胞定向迁移所必需的。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置，从而实现生物激活。到目前为止，大多数关于脂筏的存在和作用的证据都来自于间接的方法，如质膜的洗涤剂提取。由于与荧光标记相关的困难，已证明在活细胞中很难看到木筏。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。我们建议通过开发一种使用相干反斯托克斯拉曼散射(CARS)显微镜的原位微创可视化脂筏的专用仪器来弥合这一技术和信息鸿沟。CARS显微镜是一项成熟的技术，它使用两个激光光源以光谱方式确定样品的化学成分。在汽车成像中，两个光源在样本中相互作用，产生用于产生对比度图像的信号。只要激光器能够发射激发分子振动所需的波长，就可以从任何样品中产生信号。此外，在CARS成像中不需要化学荧光标记，这减少了样品的扰动。因此，CARS显微镜非常适合于原位显示脂筏结构。目前用于汽车成像的技术价格昂贵，维护性强，操作困难。通过利用光子学技术的最新发展，开发一种用于传统和多路CARS显微镜的简化和低成本的工作站，我们将在不影响细胞活力的情况下对脂筏和其他结构进行光谱成像。

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.