Super-Multiplexed Molecular Sensing in Live Cells

同时可视化具有高时空分辨率的大量不同分子物种的能力对于询问复杂和动态的生物系统至关重要，但仍然是生物成像的主要挑战。这尤其适用于细胞分析物的多重传感，包括活性物质、金属离子和大量难以标记的代谢物。由于“色障”，普遍使用的荧光显微镜在这方面受到严重限制。非线性拉曼成像技术，如受激拉曼散射（SRS），已经成为越来越有价值的生物分析工具，它提供了基于拉曼散射带较窄的谱线宽度的更多可分辨的“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变化的检测，限制了SRS的应用，主要用于获取静态图片。

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.