Super-multiplex vibrational imaging in living cells

活细胞中的超多重振动成像

基本信息

批准号：
10163876
负责人：
Wei Min
金额：
$ 31.23万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
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/10163876
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 detection sensitivity 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

项目摘要

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.

概括生物系统的组织和功能本质上是复杂且相互关联的通过一系列涉及多个交互组件的分层网络。同时可视化活细胞内大量不同的分子种类对于我们全面理解这些生物过程来说，变得不可或缺。进入系统生物学时代，这种超多重成像能力将具有变革性跨越各个领域，包括揭示神经系统的结构与功能关系；了解肿瘤异质性；研究细胞过程中的大分子编排调节，以及揭示活细胞各种细胞器之间复杂的相互作用。该项目的目标是开发通用的超多重光学显微镜平台用于同时对大量（超过 20 个）内部特定分子目标进行成像活细胞，这是传统方法的一个重要但难以实现的目标，例如为此，我们建议耦合新兴的电子预共振激发。拉曼散射 (epr-SRS) 显微镜，提供纳摩尔级检测灵敏度和窄化学特异性，具有由三键共轭光组成的新型振动探针第一代技术最近已发表，展示了一种生物系统中 24 色成像的记录（L. Wei … W. Min. Nature, 544, 465, 2017）。面向下一代技术，我们制定了系统的计划如何将这一概念具体化为一个更强大的平台，以实现高速、高灵敏度，活细胞中特定蛋白质和细胞器的超多重振动成像。我们建议建造新的显微镜仪器以显着提高成像效果速度提高几个数量级（具体目标 1），并设计新型 epr-SRS 振动探头扩展了调色板、卓越的检测灵敏度、细胞器靶向特异性和对特定蛋白质的遗传编码性（具体目标 2）伴随着这些技术。进展，然后我们将应用它来探测多个细胞器内的系统级相互作用胞质分裂和细胞凋亡动态过程中的蛋白质（具体目标 3）。如果成功实施，我们将建立一个变革性的成像平台允许研究人员研究活细胞中前所未有的大量生物分子具有卓越的灵敏度、靶向特异性、标记多功能性和生物相容性。由此产生的超多重光学显微镜将在解开复杂的问题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。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Super-multiplex imaging of cellular dynamics and heterogeneity by integrated stimulated Raman and fluorescence microscopy.

通过集成受激拉曼和荧光显微镜对细胞动力学和异质性进行超多重成像。

DOI：
发表时间：
2021-08-20
期刊：
影响因子：
5.8
作者：
Shou, Jingwen;Oda, Robert;Hu, Fanghao;Karasawa, Keiko;Nuriya, Mutsuo;Yasui, Masato;Shiramizu, Bruce;Min, Wei;Ozeki, Yasuyuki
通讯作者：
Ozeki, Yasuyuki

Metabolic Activity Phenotyping of Single Cells with Multiplexed Vibrational Probes.

使用多重振动探针对单细胞的代谢活性表型进行分析。