Creating super-scattering Raman-active genetically encoded proteins

使用光学显微镜和光谱学在真实的时间内原位研究复杂分子相互作用的能力彻底改变了我们对物理和生命科学的理解。在生物成像的背景下，光学显微镜仍然是在活细胞和组织内获得高空间和时间分辨率的唯一实用手段。荧光显微术是一种广泛使用的方法，其中荧光“标签”附着在感兴趣的生物分子上，并提供高对比度和特异性。然而，荧光染料不能容易地以非侵入性方式附着到活细胞内的特定靶分子上。这种限制在很大程度上被荧光蛋白的发现所克服，荧光蛋白可以与特定的靶蛋白基因融合。这一发现彻底改变了生物成像，并在2008年获得诺贝尔化学奖。尽管荧光显微镜是几乎任何细胞成像应用的首选方法，但它仍有一些主要缺点。首先，所有的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，会淬灭所发射的荧光强度。光漂白严重限制了作为时间函数的观察，并且通常伴随着破坏活细胞的毒性作用。此外，有机荧光团的发射光谱相当宽。这产生了一个“颜色屏障”，限制了可区分的荧光探针和相应的生物分子的数量，通常为约5个。然而，直接可视化细胞内许多不同分子种类的能力对于理解复杂系统和过程越来越重要。例如，在许多癌症类型中失调的信号通路通常涉及>50种蛋白质组分，而这些组分不可能用现有技术同时追踪。作为荧光的补充，基于拉曼散射的振动显微镜提供了光稳定性和光谱窄带。拉曼散射可以被认为是光与振动分子的非弹性碰撞。入射光子和散射光子之间的能量差等于分子获得或损失的振动能量。然而，一个主要的缺点是探测中的光子通量非常低。因此，常规的拉曼显微光谱学需要长的积分时间和/或大的入射功率，这通常与活细胞成像不兼容。为了克服这些限制，在这个项目中，我们将创造新的分子，它将拉曼散射光非常强烈，并将表现出尖锐的拉曼共振，这将使前所未有的多色成像。我们将通过工程蛋白质来实现这一目标，使其包含遗传编码的非天然拉曼活性化学键，与发色团偶联。这样，当入射光的频率接近发色团中电子吸收的频率时，拉曼散射光将有巨大的增加。重要的是，通过遗传编码，我们的新分子可以很容易地与天然蛋白质融合，从而为活细胞中的生物成像提供了一类新的光稳定标签。此外，我们将利用相干非线性增强，当两个入射激光场被用来驱动分子振动通过他们的拍音。结果，焦体积内给定类型的所有振动模式被相干驱动以同步振荡，并且拉曼散射光相长干涉。这种相干拉曼散射使用适合于活体样本中的深穿透的近红外光，并且受益于固有的3D光学切片。这一发展有可能通过提供具有上级光稳定性、多色能力、穿透深度和通过遗传编码的高度靶向分子特异性的探针和成像方法来改变活细胞显微镜领域。

The ability to study complex molecular interactions in situ in real time using light microscopy and spectroscopy has revolutionised our understanding across the physical and life sciences. In the context of bioimaging, light microscopy is still the only practical means of obtaining high spatial and temporal resolution within living cells and tissues. Fluorescence microscopy is a widely utilised method, whereby fluorescent 'tags' are attached to biomolecules of interest and provide high contrast and specificity. However, fluorescent dyes cannot easily be attached to a specific target molecule in a non-invasive manner inside a living cell. This limitation was largely overcome by the discovery of fluorescent proteins which can be genetically fused to a specific target protein. This discovery has revolutionised bioimaging and was recognised by the Nobel prize in Chemistry in 2008.Despite being the method of choice in virtually any cell imaging application, fluorescence microscopy has some major drawbacks. Firstly, all organic fluorophores are prone to photobleaching, an irreversible photo-chemical degradation process quenching the emitted fluorescence intensity. Photobleaching severely limits observations as a function of time and is often accompanied by toxic effects damaging living cells. Moreover, the emission spectrum of organic fluorophores is quite broad. This generates a "colour barrier" that limits the number of distinguishable fluorescent probes, and corresponding biomolecules, typically to about five. Yet, the ability to directly visualize many distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. For example, signalling pathways which are dysregulated in many cancer types typically involve >50 protein components, and these are impossible to track simultaneously with current techniques.Complementary to fluorescence, vibrational microscopy based on Raman scattering offers photostability and spectrally narrow bands. Raman scattering can be regarded as an inelastic collision of light with a vibrating molecule. The energy difference between the incident and scattered photon equates to the vibrational energy gained or lost by the molecule. A major drawback, however, is that photon fluxes in detection are extremely low. As a result, conventional Raman micro-spectroscopy requires long integration times and/or large incident powers, often incompatible with live cell imaging. To overcome these limitations, in this project, we will create new molecules which will Raman scatter light extremely strongly and will exhibit sharp Raman resonances that will enable unprecedented multi-colour imaging. We will achieve this by engineering proteins to contain genetically encoded non-natural Raman-active chemical bonds coupled to chromophores. In this way, there will be a huge increase of Raman scattered light when the frequency of the incident light is close to that of the electronic absorption in the chromophore. Importantly, via genetic encoding, our new molecules can be easily fused to natural proteins, thus providing a new class of photostable tags for bioimaging in living cells. Moreover, we will exploit the coherent nonlinear enhancement that is achieved when two incident laser fields are used to drive a molecular vibration via their beat note. As a result, all vibrational modes of a given type within the focal volume are coherently driven to oscillate in sync, and the Raman scattered light constructively interferes. Such coherent Raman scattering uses near-IR light suitable for deep penetration in living specimens and benefits from an intrinsic 3D optical sectioning. This development has the potential to transform the field of live cell microscopy by providing probes and imaging methods with superior photo-stability, multi-colour capabilities, penetration depth, and highly targeted molecular specificity via genetic encoding.