Label-free spatially-resolved molecular analysis of lipid bilayers by Raman spectroscopy: Going beyond the diffraction limit

在过去的二十年中，已经变得明显的是，形成细胞膜的脂质双层在许多生物过程（细胞信号传导、细胞死亡等）以及病理状况（阿尔茨海默病、心血管疾病等）中起关键作用。在分子水平上理解双层的性质并将分子机制与细胞的功能相关联对于促进我们对细胞生物学的理解以及开发针对许多疾病的新靶向疗法至关重要。尽管在这一领域的努力越来越多，但膜的许多物理和化学性质及其与其他细胞分子（例如膜受体）的相互作用仍然没有得到很好的理解，特别是在纳米级。支持的脂质双层提供了细胞膜的生物学相关模型，其提供了对双层的组成和环境的控制，以便给出膜的简化表示。然而，理解这种纳米结构的分子特性需要具有高灵敏度的先进工具，能够在不干扰系统的情况下提供详细的分子信息，并且理想地具有纳米空间分辨率。非侵入性技术也将是有利的，因为它们允许动态分子事件的测量。然而，上述要求对生命科学家目前可用的工具提出了巨大的挑战。在这个项目中提出的技术，尖端增强拉曼光谱（TERS），结合了拉曼激光光谱的化学特异性与扫描探针显微镜的高灵敏度和纳米级的空间分辨率，使收购的空间分辨的信息，从区域的膜中的各种成分分离。TERS最近已被应用于生物材料的研究，但TERS的一个关键限制已经出现：再现性。由于很难获得性能可预测的TERS提示，因此，全世界只有少数几个团体能够获得TERS。一个小组报告的结果往往不能得到其他小组的证实，特别是对于生物样本。需要采取一种新的创新办法来释放TERS的潜力，以造福于生物科学领域广泛的基础和应用研究界。我们的目标是使TERS成为一种“标准”技术，而不是少数专业实验室的储备。为了实现这一目标，我们提出了一种逐步改变的方法来设计TERS尖端，用原位方法取代传统的离线制造方法，这保证了更高水平的控制，优化和再现性（原位技术降低了处理过程中污染和损坏的风险）。与离线热蒸发方法相比，我们的初步实验表明，可以通过调整几个实验参数来控制针尖的性能。虽然我们已经证明了使用这种新技术来测量生物纳米材料的TERS光谱的能力（灵敏度提高了约10，000倍，空间分辨率提高了约20 nm），但决定TERS尖端特性的因素仍然需要优化。在这个项目中，我们将优化尖端的制造方法，然后展示使用这种新技术来研究支持的脂质双层的分子性质的可行性。我们将专注于两个和三个脂质类型组成的脂质双层中的相分离的结构域中的脂质分子的无标记映射。虽然目前的项目集中在脂质双层，但所提出的技术可用于解决生物科学中的广泛应用，例如肽纳米管，淀粉样纤维，管状蛋白或病毒轴蛋白的自组装

During the last two decades, it has become evident that the lipid bilayer forming the membranes of the cells plays a key role in many biological processes (cell signaling, cell death, etc) as well as pathological conditions (Alzheimer's disease, cardiovascular disorders, etc). Understanding the properties of the bilayers at a molecular level and correlating the molecular mechanisms with the functionality of the cells is crucial for advancing our understanding of the cell biology as well as developing new targeted therapies for many diseases. Despite increasing efforts in this field, many physical and chemical properties of the membranes and their interactions with other cellular molecules (e.g. membrane receptors) are still not well understood, especially at the nanoscale. Supported lipid bilayers provide a biologically relevant model for cell membranes, which provide control on the composition and environment of the bilayers in order to give a simplified representation of the membranes. However, understaning the molecular properties of such nanometric structures require advanced tools with a high level of sensitivity, ability to provide detailed molecular information without disturbing the system and ideally with a nanometric spatial resolution. Non-invasive techniques would also be advantageous as they allow measurements of dynamic molecular events. However, the above requirements raise huge challanges for the tools currently available to life scientists. The rechnique proposed in this project, tip-enhanced Raman spectroscopy (TERS), combines the chemical specificity of Raman laser spectroscopy with the high sensitivity and nanoscale spatial resolution of scanning probe microscopy to enable aquisition of spatially-resolved information from regions of the membranes where various constituents segregate. TERS has been applied recently to investigations of biological materials, but a key limitation of TERS has emerged: reproducibility. Difficulties in obtaining TERS tips with predictable performance have limited the availability of TERS to only few groups worldwide. It is also often that results reported by one group cannot be confirmed by other groups, especially for biological samples. A new and innovative approach is required to release the potential of TERS for the benefit of the wide ranging fundamental and applied research community in biosciences. Our aim is to make TERS a 'standard' technique and not the reserve of a few specialist laboratories.To achieve this aim, we have proposed a step-change approach to engineer TERS-tips by replacing the conventional off-line fabrication methods with an in-situ method, which promises a higher level of control, optimisation and reproducibility (in-situ techniques reduce the risk of contamination and damage during handling). Compared to the thermal evaporation off-line methods, our preliminary experiments show that the properties of the tips can be control by adjusting several experimental parameters. While we have demonstrated the ability to use this novel technique to measure TERS spectra of biological nanomaterials (~10,000 fold increase in sensitivity and ~20nm spatial resolution), the factors which determine the properties of the TERS tips still require optimisation. In this project we will optimise the tip fabrication methods and then show the feasability of using this new technique to study molecular properties of supported lipid bilayers. We will focus on label-free mapping of lipid molecules in phase-separated domains in lipid bilayers consisting of two and three lipid types. While the current project focuses on lipid bilayers, the proposed technique may be used to address a broad range of applications in biosciences, such as self-assembly of peptide nanotubes, amyloid-like fibrils, tubular proteins or virus shaft protein