Novel coherent multiphoton microscopy of living cells with nanodiamonds

本研究的目的是开发一种新的成像方式，克服目前可用的观察活细胞方法的一些严重限制。光学显微镜是细胞生物学研究中不可缺少的工具，人们不断努力开发性能更好的新技术。可以区分两种主要方法：无标签显微镜技术与依赖光学标签的成像方法。无标记技术的主要优点是观察未染色的细胞和亚细胞结构，而不需要标记过程中的伪影。相干反斯托克斯拉曼散射（CARS）近年来成为一种基于生物分子固有振动来区分生物分子的强大的无标记方法。然而，CARS的好处依赖于大量相同键的建设性干扰，因此到目前为止，在区分活细胞中的浓缩脂质方面主要是成功的。为了以高灵敏度、特异性和与活细胞成像兼容的速度可视化蛋白质和DNA，光学标记仍然是唯一的选择。在这方面，最广泛使用的标签是荧光有机染料或荧光蛋白。然而，所有有机荧光团都容易发生光漂白，这是一种不可逆的光化学降解过程，严重限制了长时间的过程观察，并伴有细胞毒性作用。作为有机荧光团的替代品，固态无机纳米颗粒作为光学标签在追求卓越的光稳定性和降低毒性方面具有很大的前景。近年来，纳米金刚石（NDs）因其基于含碳炸药爆炸的廉价大规模合成而受到世界范围的关注。它们提供小至几纳米的颗粒尺寸，高生物相容性和低细胞毒性以及有机化学的简单和多功能表面生物偶联，同时保持金刚石的结构完整性。它们在活细胞光学显微镜中的应用仍处于早期阶段，金刚石中氮空位（NV）中心的荧光发射获得了最有希望的结果。然而，这种方法受到在非发达国家生产NV中心的效率和成本的限制。迄今为止的报道表明，小的（<20nm） ND具有单个NV中心的概率非常低，并且靠近ND表面的NV中心不稳定。在这个项目中，我们提出了一项试点研究，以开发一种在细胞中成像纳米金刚石的新方法，这种方法不依赖于（因此不受）它们的荧光特性的限制。该方法基于nd的相干非线性光-物质相互作用响应，并且由于响应的非线性而具有优越的三维空间分辨率。我们将探讨两种nd的相干非线性：金刚石的电子共振四波混频（FWM）和振动共振CARS。长期愿景是实现一种新的成像技术，该技术将解决目前可用技术几乎无法解决的生物学和生物医学问题。作为一个例子，我们将定量跟踪纳米金刚石在活细胞内化后随时间的相干光信号。我们在没有光漂白的情况下，根据光信号强度来量化细胞内NDs数量的能力，将成为研究复杂细胞内通路的关键工具。

The purpose of this research is to develop a new imaging modality which overcomes a number of severe limitations in currently available methods to observe living cells.Optical microscopy is an indispensable tool in cell biology, and continuous effort is devoted to develop new techniques with improved performances. Two main approaches can be distinguished: Label-free microscopy techniques versus imaging methods which rely on optical labels. Label-free techniques have the major advantage of looking at unstained cellular and subcellular structures without unwanted artefacts from the labelling procedure. Coherent Antistokes Raman Scattering (CARS) has recently emerged as a powerful label-free method to distinguish biomolecules based on their intrinsic molecular vibrations. However, the benefit of CARS relies on the constructive interference from a large number of identical bonds, hence so far has been mostly successful in distinguishing concentrated lipids in living cells. In order to visualise proteins and DNA with high sensitivity, specificity and at speeds compatible with live cell imaging, optical-labelling is still the only option. In this respect, the most widely utilised labels are fluorescent organic dyes or fluorescent proteins. However, all organic fluorophores are prone to photo-bleaching, an irreversible photo-chemical degradation process severely limiting long time course observations and accompanied by cell toxicity effects. Alternative to organic fluorophores, solid state inorganic nanoparticles hold a great promise as optical labels in the quest for superior photostability and reduced toxicity. Recently, nanodiamonds (NDs) have gained world-wide attention due to their inexpensive large scale synthesis based on the detonation of carbon containing explosives. They offer particle sizes down to few nm, high biocompatibility and low cytotoxicity and the simple and versatile surface bioconjugation of organic chemistry while keeping the structural integrity of diamond. Their application in optical microscopy of living cells is still at an early stage, with most promising results having been obtained from the fluorescence emission of nitrogen vacancy (NV) centres in diamond. This method is however limited by the efficiency and costs in producing NV centres in NDs. Reports so far have shown that small (<20nm) NDs have a very low probability to have even a single NV center, and that NV centres close to the ND surface are not stable. In this project, we propose a pilot study to develop a novel way of imaging nanodiamonds in cells which does not rely on (and hence is not limited by) their fluorescence properties. The method is based on the coherent nonlinear light-matter interaction response of NDs and has the added benefit of a superior three-dimensional spatial resolution owing to the nonlinearity of the response. We will explore two types of coherent nonlinearities of NDs: electronically resonant four-wave mixing (FWM) and vibrationally resonant CARS of diamond. The long term vision is the realisation of a new imaging technology that will tackle biological and biomedical problems virtually impossible to address with currently available techniques. As an example, we will follow quantitatively the coherent optical signal of nanodiamonds over time after being internalised in living cells. Our ability to quantify the number of NDs within the cell over time based on the optical signal strength without photobleaching will be a key tool in the study of complicated intracellular pathways.