Super-multiplex vibrational imaging in living cells

活细胞中的超多重振动成像

• 批准号: 9921414
• 负责人: Wei Min
• 金额: $ 31.18万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9921414
• 关键词: Adopted; Alkynes; Amplifiers; Apoptosis; Azides; Biological Process; Biotin; Cell Nucleus; Cell membrane; Cells; Cellular biology; Chemicals; Clinical; Color; Complex; Crystallization; Cytokinesis; Detection; Development; Disease; Dyes; Endoplasmic Reticulum; Engineering; Event; Fluorescence; Generations; Genetic; Goals; Golgi Apparatus; Image; Imaging Techniques; Immunology; Label; Lasers; Light; Link; Lipids; Lysosomes; Malignant Neoplasms; Methods; Microscope; Microscopy; Mitochondria; Molecular; Molecular Target; Nature; Nervous system structure; Neurobiology; Optics; Organelles; Physiologic pulse; Process; Proteins; Publishing; Pump; Regulation; Research Personnel; Role; S-nitro-N-acetylpenicillamine; Series; Shapes; Specificity; Speed; Structural Protein; Structure-Activity Relationship; System; Systems Biology; Techniques; Technology; Testing; Time; Tumor Biology; biological systems; biomaterial compatibility; complex biological systems; design; detector; hybrid protein; imaging capabilities; imaging genetics; imaging modality; imaging platform; imaging probe; improved; instrumentation; interest; macromolecule; multiplexed imaging; nanomolar; next generation; novel; novel therapeutics; optical imaging; single molecule; tumor heterogeneity; vibration

Summary Biological systems are inherently complex and interrelated, as they organize and function through a series of hierarchical networks involving multiple interacting components. Hence, simultaneously visualizing a large number of distinct molecular species inside living cells has become indispensable for understanding these biological processes in a holistic manner. As we enter the era of systems biology, such super-multiplex imaging capability will be transformative across various fields including revealing structure–function relationships in nervous systems; understanding tumor heterogeneity; studying macromolecules choreography during cell regulation, as well as revealing intricate interactions among various organelles of living cells. The goal of this project is to develop a general super-multiplex optical microscopy platform for simultaneously imaging a large number (more than 20) of specific molecular targets inside live cells, an important but otherwise intractable goal by conventional methods such as fluorescence. To do so, we propose to couple the emerging electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy, offering nanomolar detection sensitivity and narrow chemical specificity, with novel vibrational probes consisting of triple-bond-conjugated light- absorbing dyes. The first-generation technique has been recently published, demonstrating a record of 24-color imaging in biological systems (L. Wei … W. Min. Nature, 544, 465, 2017). Moving towards the next-generation technology, we have laid out systematic plans as to how to crystallize this concept into a much more powerful platform to achieve high-speed, high- sensitivity, super-multiplex vibrational imaging of specific proteins and organelles in living cells. We propose to construct new microscope instrumentations to significantly boost the imaging speed by orders of magnitude (Specific Aim 1), and engineer novel epr-SRS vibrational probes with expanded color palette, superior detection sensitivity, organelle targeting specificity and genetic encodability to specific proteins (Specific Aim 2). Accompanied by these technical developments, we will then apply it to probe systems-level interactions within multiple organelles and proteins during dynamical processes of cytokinesis and apoptosis (Specific Aim 3). If successfully implemented, we will establish a transformative imaging platform that could allow researchers to interrogate an unprecedented large number of bio-molecules in living cells with superb sensitivity, targeting specificity, labeling versatility, and biocompatibility. The resulting super-multiplex optical microscopy would find wide applications in unraveling complex biological systems such as cell biology, neurobiology, immunology, and tumor biology.

概括 当生物系统组织和功能时，生物系统本质上是复杂且相互关联的 通过一系列分层网络涉及多个交互组件。因此， 同时可视化活细胞内部大量不同的分子物种具有 对于以整体方式理解这些生物学过程是必不可少的。像我们 输入系统生物学时代，这种超级插图成像能力将具有变革性 在各个领域，包括在神经系统中揭示结构 - 功能关系； 了解肿瘤异质性；研究细胞期间的大分子编排 调节，并揭示了各种活细胞细胞器之间的复杂相互作用。 该项目的目的是开发一个一般的超级型光学显微镜平台 为了简单地模仿内部的大量特定分子靶 活细胞，通过常规方法（例如 荧光。为此，我们提议将刺激的新兴电子预呼应搭配 拉曼散射（EPR-SRS）显微镜，提供纳摩尔检测灵敏度和狭窄 化学特异性，具有新的振动问题，包括三键偶联的光 吸收染料。第一代技术最近发表了，证明了 生物系统中24色成像的记录（L. Wei…W。W.Min。Nature，544，465，2017）。 走向下一代技术，我们制定了系统的计划 如何将这个概念结晶成一个更强大的平台，以实现高速，高速 灵敏度，活细胞中特定蛋白质和细胞器的超级振动振动成像。 我们建议构建新的显微镜仪器以显着增强成像 速度按数量级（特定的目标1）和工程师的新型EPR-SRS振动问题 随着调色板的扩展，出色的检测灵敏度，靶向特异性和 特定蛋白质的遗传编码（特定目标2）。伴随着这些技术 然后，我们将其应用于多个细胞器中的探测系统级相互作用 和蛋白质在细胞因子和凋亡的动态过程中（特定目标3）。 如果成功实施，我们将建立一个可以变革的成像平台 允许研究人员询问活细胞中空前的大量生物分子 具有出色的灵敏度，靶向特异性，标签多功能性和生物相容性。 由此产生的超级插曲光学显微镜将在解开复合物中找到广泛的应用 生物系统，例如细胞生物学，神经生物学，免疫学和肿瘤生物学。