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

超高分辨率单分子受激拉曼散射 (SRS) 显微镜

• 批准号: 9899269
• 负责人: Wei Min
• 金额: $ 31.54万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899269
• 关键词: Address; Adopted; Algorithms; Alkynes; Amino Acids; Amplifiers; Area; Basic Science; Beds; Biological; Biophotonics; Blinking; Cancer Biology; Carbon; Cells; Chemical Structure; Chemicals; Color; Computational algorithm; Coupling; Crystallization; Detection; Development; Diagnostic; Disease; Dyes; Fluorescence; Fluorescence Microscopy; Generations; Glucose; Goals; Home environment; Image; Immunology; Label; Lasers; Lipids; Medical Research; Methods; Microscope; Microscopy; Molecular Target; Neurosciences; Optics; Pharmaceutical Preparations; Property; Pump; Radial; Reporting; Resolution; Sampling; Scheme; Specificity; Speed; System; Techniques; Technology; Testing; Tubulin; base; bioimaging; biological research; biomaterial compatibility; complex biological systems; design; detector; diffraction of light; imaging platform; improved; instrument; instrumentation; interdisciplinary approach; invention; molecular scale; multiplexed imaging; next generation; novel; precision medicine; second harmonic; single molecule; vibration

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

项目概要 超分辨率光学显微镜有望彻底改变生物成像，因为它使非 分子尺度的侵入性询问。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由于使用 荧光作为对比机制。特别是，它具有三个主要局限性：（1）它不能揭示化学成分。 样品的成分； (2) 由于荧光相对较大，因此无法询问小生物分子 标签； (3)由于色差的原因，无法对大量目标进行成像（只能对2~5种荧光颜色进行成像） 实际解决了）。 该项目的目标是通过利用受激光来开发一种新型的超分辨率成像平台 拉曼散射（SRS）作为对比机制。自2008年发明以来，SRS在过去的10年里 显微镜在生物医学成像领域产生了广泛的影响。作为一种化学敏感方法，SRS 是 以其定量方式的无标记化学分析而闻名。随着近年来微型化的发展 生物正交标签，例如炔烃，SRS 已被证明可以成功地询问多种 小生物分子，例如脂质、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级多重电子预共振 (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 是对流行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 解决。 （1）如何提高普通化学成像和生物小分子成像的分辨率； (2) 如何突破超多重epr-SRS成像的衍射极限； (3)如何制定配套 用于单分子 SRS 的振动染料。 为了实现这些目标，我们制定了一个系统的计划，如何将这个概念具体化为一个 强大的技术平台。已计划采用跨学科方法，包括仪器仪表 开发、计算成像和新型探针合成。在目标 1 中，我们将开发和建立新的 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—— 生成振动探头。如果成功实施，我们将建立一个变革性的平台。这 由此产生的超分辨率化学成像将在系统地揭示复杂的问题上得到广泛的应用 生物系统，例如用于基础研究、疾病的神经科学、免疫学和癌症生物学 诊断和精准医学。 1