Super-multiplex vibrational imaging in living cells

总结 生物系统在组织和运作时， 通过一系列涉及多个交互组件的分层网络。因此，我们认为， 同时观察活细胞内大量不同的分子种类， 对于全面理解这些生物过程是不可或缺的。正如我们 一旦进入系统生物学时代，这种超多路复用成像能力将是革命性的 跨各个领域，包括揭示神经系统中的结构-功能关系; 了解肿瘤异质性;研究细胞周期中的大分子编排 调节，以及揭示活细胞的各种细胞器之间的复杂相互作用。 本计画的目标是发展一个通用的超多重光学显微镜平台 用于同时成像内部的大量（超过20个）特定分子目标， 活细胞，这是一个重要但难以通过常规方法实现的目标， 荧光。为此，我们建议将新出现的电子预共振激发 拉曼散射（epr-SRS）显微镜，提供纳摩尔检测灵敏度和窄 化学特异性，与新的振动探针组成的三键共轭轻， 吸收染料。第一代技术最近发表，展示了一种 生物系统中的24色成像记录（L.威...威。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。 我们建议构建新的显微镜仪器，以显着提高成像 速度的数量级（具体目标1），并设计新的epr-SRS振动探针 具有扩展的调色板、上级检测灵敏度、细胞器靶向特异性， 特定蛋白质的遗传编码能力（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水平的相互作用 以及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， 使研究人员能够在活细胞中询问前所未有的大量生物分子， 具有极好的灵敏度、靶向特异性、标记通用性和生物相容性。的 由此产生的超多路光学显微镜将在解开复杂的 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.