Super-multiplex vibrational imaging in living cells

摘要 生物系统在组织和运行时，本质上是复杂和相互关联的 通过涉及多个交互组件的一系列分层网络。因此， 同时观察活细胞内大量不同的分子物种 成为以整体方式理解这些生物过程不可或缺的一部分。因为我们 进入系统生物学时代，这种超多路成像能力将是变革性的 横跨多个领域，包括揭示神经系统的结构-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，以及揭示活细胞的各种细胞器之间错综复杂的相互作用。 本课题的目标是开发一个通用的超多路光学显微镜平台 用于同时成像大量(20多个)内部的特定分子目标 活细胞，这是一个重要但在其他方面难以通过常规方法实现的目标，例如 荧光。为了做到这一点，我们建议将新出现的电子预共振激励 拉曼散射(EPR-SRS)显微镜，提供纳米分子检测灵敏度和窄 化学专一性，具有由三键共轭光组成的新型振动探针- 吸收染料。第一代技术最近发布，展示了一种 生物系统中的24色成像记录(L.魏…W.Min.《自然》，544,465,2017)。 迈向下一代科技，我们已制定有系统的计划，包括 如何将这一概念具体化为一个功能更强大的平台，实现高速、高速、 活细胞中特定蛋白质和细胞器的灵敏度、超多路振动成像。 我们建议建造新的显微镜仪器来显著提高成像 速度达数量级(具体目标1)，设计新型EPR-SRS振动探头 具有扩展的调色板、卓越的检测灵敏度、细胞器靶向性和 特定蛋白质的遗传可编码性(特定目标2)。伴随着这些技术 发展，然后我们将应用它来探测多个细胞器内的系统级别的相互作用 和蛋白质在胞质分裂和凋亡的动态过程中(特定目标3)。 如果成功实施，我们将建立一个变革性的成像平台，可以 允许研究人员在活细胞中询问前所未有的大量生物分子 具有极高的敏感性、靶向性、标记的多功能性和生物兼容性。这个 由此产生的超多重光学显微镜将在解开复杂结构方面获得广泛的应用 生物系统，如细胞生物学、神经生物学、免疫学和肿瘤生物学。

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.