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Novel coherent multiphoton microscopy of living cells with nanodiamonds

使用纳米金刚石进行活细胞的新型相干多光子显微镜

基本信息

批准号：
BB/J021008/1
负责人：
Paola Borri
金额：
$ 15.16万
依托单位：
CARDIFF UNIVERSITY
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2012
资助国家：
英国
起止时间：
2012 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FJ021008%2F1
关键词：
Novel coherent multiphoton microscopy living

项目摘要

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.

本研究的目的是开发一种新的成像模式，以克服现有方法在观察活细胞方面的一些严重限制。光学显微镜是细胞生物学中不可或缺的工具，并不断致力于开发性能更好的新技术。可以区分两种主要的方法：无标记显微镜技术和依赖于光学标记的成像方法。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，而不会出现标记过程中不需要的伪影。相干反斯托克斯拉曼散射(CARS)是最近出现的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大多是成功的。为了以高灵敏度、高特异度和与活细胞成像兼容的速度显示蛋白质和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，这是一个不可逆的光化学降解过程，严重限制了长时间的观察，并伴随着细胞毒性效应。作为有机荧光团的替代品，固态无机纳米颗粒在寻求更好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)以其廉价的、基于含碳炸药爆炸的大规模合成而受到世界各国的广泛关注。它们提供的颗粒尺寸小至几纳米、高生物兼容性和低细胞毒性，以及简单和多功能的有机化学表面生物偶联，同时保持了钻石的结构完整性。它们在活细胞光学显微镜中的应用仍处于早期阶段，最有希望的结果是通过钻石中氮空位(NV)中心的荧光发射而获得的。然而，这种方法受到在NDS生产NV中心的效率和成本的限制。到目前为止的报告已经表明，小的(&lt；20 nm)NDS甚至只有一个NV中心的可能性非常低，并且靠近ND表面的NV中心不稳定。在这个项目中，我们提出了一项初步研究，以开发一种在细胞中成像纳米钻石的新方法，这种方法不依赖于(因此也不受)它们的荧光特性。该方法基于NDS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，具有更高的三维空间分辨率。我们将探索NDS的两种相干非线性：电子共振四波混频(FWM)和钻石的振动共振CARS。长远的愿景是实现一种新的成像技术，该技术将解决目前可用的技术几乎不可能解决的生物和生物医学问题。例如，我们将定量跟踪纳米钻石在活细胞中内化后随时间的相干光学信号。我们能够在没有光漂白的情况下根据光信号强度来量化细胞内NDS的数量，这将是研究复杂的细胞内途径的关键工具。