Ultrahigh-resolution and single-molecule stimulated Raman scattering (SRS) microscopy

项目总结 超分辨率光学显微镜有望给生物成像带来革命性的变化，因为它使非 分子规模的侵入性审讯。事实上，超分辨率荧光显微镜的出现 迅速影响了生物学家研究细胞和亚细胞现象的方式。 然而，超分辨率荧光显微镜有根本的局限性，因为使用 荧光作为对比机制。特别是，它有三大局限性：(1)它不能揭示化学物质 样品的组成；(2)由于相对较大的荧光，它不能询问小的生物分子 标签；(3)由于颜色障碍，无法对大量目标成像(只能有2~5种荧光颜色 实际上解决了问题)。 本项目的目标是开发一种新型的超分辨率成像平台，利用受激波 拉曼散射(SRS)作为对比机制。自2008年发明以来，在过去的10年里，SRS 显微技术在生物医学成像领域产生了广泛的影响。作为一种化学敏感的方法，SRS是 以其无标记的定量化学分析而闻名。随着微型机的最新发展 生物正交标签，如乙炔，SRS已被证明成功地询问了广泛的光谱 小的生物分子，如脂质、葡萄糖、氨基酸和药物。最近，新型振动染料 据报道，具有精细光谱分辨率的调色板可以实现超多路电子预共振 (EPR)同时对20多个目标进行SRS成像。重要的是，SRS显微镜的所有这些用途都是 受光衍射的限制。 由于SRS是对普遍荧光的完美补充对比机制， 目前的提案旨在开发必要的方法，将SRS显微镜带入超级领域 决议。(1)如何提高普通化学成像和小分子生物成像的分辨率； 如何突破超多路EPR-SRS成像的衍射极限；(3)如何发展匹配 用于单分子SRS的振动染料。 为了实现这些目标，我们制定了一项有系统的计划，如何将这一概念具体化为 强大的技术平台。已经计划了一种跨学科的方法，包括仪器 开发、计算成像和新型探针合成。在目标1中，我们将开发和建设新的 仪器仪表。在目标2中，我们将探索新的计算算法。在目标3中，我们将设计下一步- 产生振动探头。如果成功实施，我们将建立一个变革性的平台。这个 由此产生的超分辨率化学成像将在系统地解开复杂结构方面得到广泛的应用 基础研究、疾病研究的神经科学、免疫学和癌症生物学等生物系统 诊断学和精准医学。 1

Project summary Super-resolution optical microscopy promises to revolutionize biological imaging, as it enables non- invasive interrogation at molecular scale. Indeed, the emergence of super-resolution fluorescence microscopy has quickly impacted the way biologists study cells and subcellular phenomenon. However, super-resolution fluorescence microscopy has fundamental limitations due to the use the fluorescence as contrast mechanism. In particular, it has three major limitations: (1) it cannot reveal chemical composition of the sample; (2) it cannot interrogate small biomolecules due to the relatively bulky fluorescent tags; (3) it cannot image a large number of targets due to the color barrier (only 2~5 fluorescent colors can be practically resolved). The goal of this project is to develop a novel super-resolution imaging platform by exploiting stimulated Raman scattering (SRS) as the contrast mechanism. During the past 10 years since its invention in 2008, SRS microscopy has made widespread impact in biomedical imaging. Being a chemically sensitive method, SRS is well known for its label-free chemical analysis in a quantitative manner. With the recent development of tiny bio-orthogonal tags such as alkynes, SRS has been proven successful in interrogating a wide spectrum of small biomolecules such as lipids, glucose, amino acids, and drugs. Very recently, novel vibrational dye palettes with fine spectral resolution have been reported to achieve super-multiplex electronic pre-resonance (epr) SRS imaging of more than 20 targets simultaneously. Importantly, all these utilizes of SRS microscopy is limited by light diffraction. With SRS being a perfectly complementary contrast mechanism to the prevalent fluorescence, the current proposal aims to develop the necessary methods to bring SRS microscopy to the realm of super resolution. (1) How to improve the resolution for general chemical imaging and small biomolecule imaging; (2) how to break the diffraction limit of the super-multiplex epr-SRS imaging; (3) how to develop the matching vibrational dyes for single molecule SRS. Towards these goals, we had laid out a systematic plan as to how to crystallize this concept into a powerful technology platform. An inter-disciplinary approach has been planned out including instrumentation development, computational imaging, and novel probes synthesis. In Aim 1, we will develop and build new instrumentation. In Aim 2, we will explore new computational algorithm. In Aim 3, we will design next- generation vibrational probes. If successfully implemented, we will establish a transformative platform. The resulting super-resolution chemical imaging would find wide applications in systematically unraveling complex biological systems such as neuroscience, immunology and cancer biology for basic research, disease diagnostics and precision medicine. 1