Nonlinear plasmonic biosensing and functional imaging

对EPSRC研究领导人的额外支持将用于资助两个相互关联的工作，这些工作与当前的领导奖学金协同作用，同时扩展到新的方向。这项工作将集中在一个新的途径使用的相干光学非线性（称为四波混频- FWM）对金属纳米粒子（NPs）的生物传感单分子直接在细胞内，并在FWM与电子显微镜的组合作为一个新的相关光电子显微镜（CLEM）的方法。为了深入定量了解细胞功能，生物学问题越来越多地转移到单分子水平，并要求能够测量单个生物分子的动态行为，同时直接在细胞内进行构象变化和结合事件的技术。用于这些单分子研究的光学技术通常使用有机荧光团作为光学标记，然而其遭受漂白和不可逆降解。金属纳米颗粒作为替代标签越来越受到关注，因为它们有效地吸收和散射特定波长的光（称为局部表面等离子体共振- LSPR）并且不会闪烁或光漂白。重要的是，LSPR波长对紧邻的另一金属NP的存在非常敏感，这取决于颗粒间距离。因此，通过测量在结合和形成NP二聚体时发生的LSPR位移，连接到生物分子的金属NP可以用作具有单分子灵敏度的结合事件的“等离子体标尺”。然而，迄今为止报道的所有实验都未能证明细胞内具有小NP二聚体的生物传感，因为它们依赖于不是无背景的光学方法。在我们的实验室中，我们最近开发了一种新的FWM技术，即使在高度散射和荧光的环境中，也能够分辨单个小的（<40 nm）金属纳米粒子，并且在非常低的功率下工作，因此与活细胞成像兼容。在这个项目中，我们将率先使用这种新的FWM检测与NP二聚体的局部生物传感与单分子灵敏度直接在细胞内。相关的研究链将结合联合收割机FWM与电子显微镜（EM）。这将通过解析NP二聚体来支持生物传感链，并且由于EM的亚纳米空间分辨率而能够精确测量它们的颗粒间距离。此外，CLEM是了解细胞内过程的独特工具。例如，细胞必须制造新的蛋白质，并将其运输到正确的位置以正常发挥功能。同样，细胞必须解释来自外部的信号并正确地传递它们。用什么载体来传递这些信号？蛋白质在哪里以及如何聚集或分离？虽然动力学可以通过光学显微镜进行研究，但其有限的分辨率（> 100 nm）需要EM进行精确定位，但EM仅提供静态图像。在CLEM中，用标记物标记细胞，并且首先用光学显微镜在活细胞中跟踪细胞内事件。当观察到感兴趣的事件时，细胞被快速固定并进行EM处理。目前的方法使用用于光学显微镜的荧光团与用于EM的金NP缀合。然而，荧光团光漂白，并且当与金属NP缀合时可能经历淬灭。此外，有一个未知的修改之间的最后一个图像从光学显微镜和固定的细胞在EM研究，因为EM处理程序通常会破坏荧光团。我们将克服这些局限性，使用我们的新的FWM检测作为光学显微镜在CLEM过程中。FWM直接可视化裸露的金属纳米颗粒，而没有荧光团，并且具有高度的光稳定性和无背景。该项目的高潮将是两个研究链的结合，使生物传感CLEM。

This additional support for EPSRC Research Leaders will be used to fund two interrelated strands of work which synergise with the current Leadership fellowship whilst expanding into new directions. The work will focus on a new avenue of using the coherent optical nonlinearity (called Four-Wave Mixing - FWM) of pairs of metallic nanoparticles (NPs) for biosensing single molecules directly inside cells, and on the combination of FWM with electron microscopy as a new correlative light electron microscopy (CLEM) approach. For an in depth quantitative understanding of cellular functions, biological questions are increasingly moving to the single molecule level and demanding techniques able to measure the dynamic behaviour of single biomolecules while undergoing conformational changes and binding events directly inside cells. Optical techniques for these single molecule studies typically use organic fluorophores as optical labels, which however suffer from bleaching and irreversible degradations. Metallic NPs have attracted increasing attention as alternative labels since they efficiently absorb and scatter light at specific wavelengths (called localised surface plasmon resonances - LSPR) and do not blink or photobleach. Importantly, the LSPR wavelength is very sensitive to the presence of another metallic NP in close proximity depending on the inter-particle distance. Metallic NPs attached to biomolecules can thus be used as "Plasmon rulers" of binding events with single molecule sensitivity by measuring the LSPR shift which occurs upon binding and formation of a NP dimer. However, all experiments reported to date failed to demonstrate biosensing with small NP dimers inside cells, since they rely on optical methods which are not background-free. In our laboratory, we have recently developed a novel FWM technique capable of resolving single small (< 40nm) metallic NPs background-free even in a highly scattering and fluorescing environment and operating at very low powers, hence compatible with live cell imaging. In this project we will pioneer the use of this novel FWM detection with NP dimers for local biosensing with single molecule sensitivity directly inside cells. The interrelated research strand will combine FWM with electron microscopy (EM). This will support the biosensing strand by resolving NP dimers and enabling accurate measurement of their inter-particle distance owing to the sub-nanometer spatial resolution of EM. Moreover, CLEM is a unique tool to understand intracellular processes. For instance, cells have to make new proteins and transport these to the correct places to function properly. Likewise cells have to interpret signals from the outside and route them correctly. What carriers are being used to convey these signals? Where and how do proteins aggregate or segregate? Although dynamics can be studied by light microscopy, its limited resolution (>100nm) requires EM for precise localisation, but EM alone gives only a static image. In CLEM cells are labelled with markers and intracellular events are first followed in living cells with light microscopy. When an interesting event is observed, cells are rapidly fixed and processed for EM. Current approaches use fluorophores for light microscopy conjugated to gold NPs for EM. However, fluorophores photobleach, and when conjugated to metallic NPs might undergo quenching. In addition, there is an unknown modification between the last image from the light microscope and the fixed cell studied in EM since the EM processing procedures usually destroy the fluorophore. We will overcome these limitations by using our novel FWM detection as light microscopy in the CLEM process. FWM directly visualises bare metallic NPs without fluorophores, and is highly photostable and background-free. The culmination of this project will be the combination of both research strands to enable biosensing CLEM.