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Shedding new light on cells with coherent multiphoton nanoscopy

通过相干多光子纳米显微镜为细胞提供新的线索

基本信息

批准号：
EP/I005072/1
负责人：
Paola Borri
金额：
$ 146.46万
依托单位：
CARDIFF UNIVERSITY
依托单位国家：
英国
项目类别：
Fellowship
财政年份：
2010
资助国家：
英国
起止时间：
2010 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=EP%2FI005072%2F1
关键词：
Shedding new light cells coherent

项目摘要

The aim of this research is the realization of a novel imaging modality to enable the observation of living cells and tissues under physiological conditions with unprecedented sensitivity and spatial resolution, without the need to stain them with fluorophores. The technique, based on the interaction of light with matter in the coherent regime, will feature a unique combination two process: Coherent Antistokes Raman Scattering (CARS) of biomolecules in living cells and Four-Wave Mixing (FWM) imaging of metallic nanoparticles (NPs). This technology will progress the field of optical 'nanoscopy', advance our understanding in physics and material sciences, tackle biological problems that are virtually impossible to address with currently available techniques, and will be of relevance in medical applications to improve the diagnostic and treatment of diseases.Optical microscopy is an indispensable tool that is driving progress in cell biology, however most cellular constituents have no colour and they are hard to distinguish under a light microscope unless they are stained. Fluorescence microscopy using organic dyes attached to biomolecules or fluorescent proteins has provided a highly sensitive method of visualizing biomolecules. However, when used for observations in living cells, these modified biomolecules raise questions if their behaviour is real or artefactual. Furthermore, all organic fluorophores are prone to photo-bleaching, an irreversible degradation of the fluorescence intensity after excitation with light, which severely limits time-course observations and is accompanied by toxicity effects and consequent cell damage. In CARS the image contrast is obtained by detecting light which is scattered by vibrating bonds in unstained biomolecules. Although this scattering phenomenon produces a very weak signal, it can be coherently enhanced when two short laser pulses are used to excite the vibrations (generating CARS) so that the scattered light from all bonds of the same type constructively interfere. However, CARS still requires a high number of molecules to achieve sufficient signal for detection, and the existence of a background severely limits its sensitivity. Another problem is the spatial resolution limited by the optical diffraction (>100nm).In this programme, I will overcome these limitations by developing a background free CARS detection combined with the light enhancement occurring in the nanoscale range near a metallic NP, to achieve nanometric spatial resolution and high sensitivity. The NP will be located and tracked using FWM imaging, recently demonstrated in our laboratory, here in a new version to enable nanometric position accuracy in all three directions. FWM detection will also report the local thermal conductivity of the NP surroundings. In addition the microscope will feature trapping of the NP with optical tweezers to position it in a region of interest and/or to measure forces applied to it.The ability to map the intrinsic chemical composition of nanoscale regions together with their thermal and mechanical properties in living cells will have a major impact in solving important biomedical problems. For example, we will determine whether cell membranes perform their function through the assembly of lipid nanodomains or 'rafts'. Their existence is thought to play a key role in basic biological functions and in many diseases (eg influenza and HIV) but is also controversial owing to their small size. Another application will be to determine the local membrane environment associated with endocytosis which is crucial, beyond fundamental biology, for the design of drug delivery and therapeutic strategies. More in general, this novel imaging modality will allow us to address biological systems where the manipulation and photo-toxicity associated with the use of fluorescence markers is unacceptable, e.g. in the areas of in-vitro fertilization and cancer research.

这项研究的目的是实现一种新的成像模式，使活细胞和组织的观察在生理条件下具有前所未有的灵敏度和空间分辨率，而不需要用荧光团染色。该技术基于光与物质在相干状态下的相互作用，将具有两个过程的独特组合：活细胞中生物分子的相干反斯托克斯拉曼散射（汽车）和金属纳米颗粒（NP）的四波混频（FWM）成像。这项技术将推动光学“纳米”领域的发展，促进我们对物理学和材料科学的理解，解决目前可用技术几乎不可能解决的生物学问题，并将在医疗应用中发挥重要作用，以改善疾病的诊断和治疗。光学显微镜是推动细胞生物学进步的不可或缺的工具，然而，大多数细胞成分没有颜色，除非染色，否则在光学显微镜下难以区分。荧光显微镜使用有机染料连接到生物分子或荧光蛋白提供了一个高度敏感的方法可视化生物分子。然而，当用于观察活细胞时，这些修饰的生物分子的行为是真实的还是人为的就产生了问题。此外，所有有机荧光团都易于发生光漂白，即在用光激发后荧光强度的不可逆降解，这严重限制了时间过程的观察，并伴随着毒性效应和随后的细胞损伤。在汽车中，图像对比度通过检测由未染色生物分子中的振动键散射的光来获得。虽然这种散射现象产生非常弱的信号，但当使用两个短激光脉冲来激发振动（产生汽车）时，它可以被相干增强，以便来自相同类型的所有键的散射光相长干涉。然而，汽车仍然需要大量的分子来获得足够的检测信号，背景的存在严重限制了其灵敏度。另一个问题是空间分辨率受到光学衍射（> 100 nm）的限制。在这个项目中，我将通过开发一种无背景汽车检测结合金属NP附近纳米尺度范围内发生的光增强来克服这些限制，以实现纳米空间分辨率和高灵敏度。NP将使用FWM成像进行定位和跟踪，最近在我们的实验室中进行了演示，这里是一个新版本，可以在所有三个方向上实现纳米级的位置精度。FWM检测还将报告NP周围环境的局部热导率。此外，显微镜将具有捕获的NP与光镊将其定位在感兴趣的区域和/或测量施加到它的力。映射的能力，连同其在活细胞中的热和机械性能的纳米级区域的内在化学成分将有重大影响，在解决重要的生物医学问题。例如，我们将确定细胞膜是否通过脂质纳米结构域或“筏”的组装来执行其功能。它们的存在被认为在基本的生物功能和许多疾病（如流感和艾滋病毒）中起着关键作用，但由于它们的小尺寸也存在争议。另一个应用将是确定与内吞作用相关的局部膜环境，这对于药物递送和治疗策略的设计是至关重要的，超出了基础生物学。更一般地说，这种新的成像模式将使我们能够解决与使用荧光标记物相关的操作和光毒性是不可接受的生物系统，例如在体外受精和癌症研究领域。