Super-Multiplexed Molecular Sensing in Live Cells

同时显示大量具有高时空分辨率的不同分子物种的能力对于询问复杂和动态的生物系统是至关重要的，但仍然是生物成像中的一个主要挑战。对于包括活性物质、金属离子和大量难以标记的代谢物在内的细胞分析物的多重传感尤其如此。由于“色障”的存在，目前流行的荧光显微镜受到了严重的限制。像受激拉曼散射(SRS)这样的非线性拉曼成像技术已经成为一种越来越有价值的生物分析工具，它基于拉曼散射带的较窄的光谱线宽提供了更多数量的可分辨颜色。然而，目前的拉曼标记技术缺乏响应性，阻碍了对感兴趣物种浓度随时间的变化的检测，限制了SRS的应用，仅限于获取主要是静态的图像。 该项目的目标是建立下一代化学工具箱和补充仪器，以实现对活细胞中瞬时物种和事件的高速、超多路复用监测，这是其他传统光学方法的一个重要但在其他方面难以实现的目标。我们建议设计和合成一个新颖的响应性振动探针库，用于离子、小反应分子和酶活性的SRS和电子预共振(EPR)-SRS传感(特定目标1)，可用于最先进的SRS显微镜仪器。这项新技术将在内质网和线粒体相互作用以及活细胞重塑的背景下对这些分子靶标的超多路可视化进行测试(特定目标2)。拟议计划的成功完成将建立一种变革性技术，能够以高时空分辨率同时、动态地显示关键分子成分、结构和过程。这种能力对于获得对细胞网络及其串扰的综合看法是必不可少的，并将在解开细胞生物学、神经生物学、免疫学和肿瘤生物学领域的复杂系统中获得广泛的应用。

The ability to visualize simultaneously a large number of distinct molecular species with high spatiotemporal resolution is crucial for interrogating complex and dynamic biological systems but still remains a major challenge in bioimaging. This is especially true for multiplex sensing of cellular analytes including reactive species, metal ions, and a plethora of difficult-to-tag metabolites. The prevalent fluorescence microscopy is severely limited for this because of the “color barrier”. Non-linear Raman imaging techniques such as Stimulated Raman Scattering (SRS) have become an increasingly valuable bioanalytical tool by offering a much greater number of resolvable ‘colors’ based on the narrower spectral linewidth of the Raman scattering bands. However, the lack of responsiveness of current Raman ‘tagging’ technologies hinders detection of changes in concentration of species of interest over time, limiting the application of SRS to the acquisition of mostly static pictures. The goal of this project is to establish the next generation chemical toolbox and complementary instrumentation to enable high-speed, super-multiplexed monitoring of transient species and events in live cells, an important but otherwise intractable goal by other traditional optical methods. We propose to design and synthesize a library of novel responsive vibrational probes for SRS and electronic pre-resonance (epr) -SRS sensing of ions, small reactive molecules and enzymatic activity (Specific Aim 1), amenable for use with state-of-the-art SRS microscopy instrumentation. The new technology will be tested for the super-multiplexed visualization of these molecular targets in the context of ER and mitochondrial interactions and remodeling in live cells (Specific Aim 2). Successful completion of the proposed plan will establish a transformative technology that would enable concurrent, dynamic visualization of key molecular components, structures and processes with high spatiotemporal resolution. This capability is essential for gaining an integrated view of cellular networks and their crosstalk and would find wide applications in unraveling complex systems in the realm of cell biology, neurobiology, immunology, and tumor biology.