Creating super-scattering Raman-active genetically encoded proteins

使用光学显微镜和光谱学在现场实时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。在生物成像方面，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。荧光显微镜是一种广泛使用的方法，通过这种方法，荧光‘标签’被贴在感兴趣的生物分子上，并提供高对比度和特异性。然而，荧光染料不容易在活细胞内以非侵入性的方式连接到特定的目标分子上。这一局限性在很大程度上被荧光蛋白的发现所克服，这种荧光蛋白可以在基因上与特定的靶蛋白融合。这一发现给生物成像带来了革命性的变化，并得到了2008年诺贝尔化学奖的认可。尽管荧光显微镜是几乎所有细胞成像应用的首选方法，但它也有一些重大缺陷。首先，所有有机荧光团都容易发生光漂白，这是一种不可逆光化学降解过程，使发射的荧光强度猝灭。光漂白严重限制了作为时间函数的观察，并经常伴随着损害活细胞的毒性效应。此外，有机荧光团的发射光谱相当宽广。这就产生了一道“颜色屏障”，将可区分的荧光探针和相应的生物分子的数量限制在通常为五个左右。然而，直接显示细胞内许多不同分子物种的能力对于理解复杂的系统和过程越来越重要。例如，在许多癌症类型中调节失调的信号通路通常涉及&gt；50蛋白质组分，而目前的技术不可能同时跟踪这些蛋白质组分。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。拉曼散射可以看作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，探测中的光子通量极低。因此，传统的拉曼显微光谱需要较长的积分时间和/或较大的入射功率，这往往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，这些分子将产生极其强烈的拉曼散射光，并将表现出尖锐的拉曼共振，从而实现前所未有的多色成像。我们将通过改造蛋白质来实现这一点，使其包含基因编码的非天然拉曼活性化学键，与生色团偶联。这样，当入射光的频率接近生色团中电子吸收的频率时，拉曼散射光就会有很大的增加。重要的是，通过基因编码，我们的新分子可以很容易地与天然蛋白质融合，从而为活细胞的生物成像提供一类新的光稳定性标签。此外，我们还将利用两个入射激光场通过节拍音符驱动分子振动时实现的相干非线性增强。结果，焦体积内给定类型的所有振动模式被相干地驱动以同步振荡，并且拉曼散射光建设性地干涉。这种相干拉曼散射使用适合于深入穿透活标本的近红外光，并得益于固有的3D光学切片。这一发展具有改变活细胞显微镜领域的潜力，通过遗传编码提供具有卓越的光稳定性、多色能力、穿透深度和高度靶向性的分子特异性的探针和成像方法。

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.