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.