Shedding new light on cells with coherent multiphoton nanoscopy

本研究的目的是实现一种新的成像方式，使在生理条件下以前所未有的灵敏度和空间分辨率观察活细胞和组织，而无需用荧光团染色。该技术基于光与物质在相干状态下的相互作用，将具有两种独特的结合过程：活细胞中生物分子的相干反斯托克斯拉曼散射（CARS）和金属纳米粒子（NPs）的四波混合（FWM）成像。这项技术将推动光学“纳米镜”领域的发展，促进我们对物理和材料科学的理解，解决目前技术几乎无法解决的生物学问题，并将在医学应用中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很难区分。荧光显微镜利用附着在生物分子或荧光蛋白上的有机染料提供了一种高度敏感的生物分子可视化方法。然而，当对活细胞进行观察时，这些经过修饰的生物分子的行为是真实的还是人为的，这就引发了问题。此外，所有有机荧光团都容易发生光漂白，即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，并伴有毒性作用和随之而来的细胞损伤。在CARS中，图像对比度是通过检测未染色生物分子中由振动键散射的光来获得的。尽管这种散射现象产生的信号非常微弱，但当使用两个短激光脉冲来激发振动（产生CARS）时，它可以被相干增强，从而使来自同一类型化学键的散射光产生建设性干涉。然而，CARS仍然需要大量的分子来获得足够的检测信号，并且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限于光学衍射（>100nm）。在这个项目中，我将通过开发一种无背景的CARS检测，结合金属NP附近纳米尺度范围内的光增强，来克服这些限制，以实现纳米空间分辨率和高灵敏度。我们将使用FWM成像技术对NP进行定位和跟踪，该技术最近在我们的实验室进行了演示，在这里的一个新版本中，可以在所有三个方向上实现纳米级定位精度。FWM检测还将报告NP环境的局部热导率。此外，显微镜将使用光学镊子捕获NP，以将其定位在感兴趣的区域和/或测量施加在其上的力。绘制纳米级区域的内在化学成分及其在活细胞中的热学和力学特性的能力将对解决重要的生物医学问题产生重大影响。例如，我们将确定细胞膜是否通过脂质纳米结构域或“筏”的组装来发挥其功能。它们的存在被认为在基本生物学功能和许多疾病（如流感和艾滋病毒）中发挥关键作用，但由于它们的体积小，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，这对药物传递和治疗策略的设计至关重要，超越基础生物学。更一般地说，这种新颖的成像方式将使我们能够解决与荧光标记使用相关的操作和光毒性不可接受的生物系统，例如在体外受精和癌症研究领域。

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.