Raman probes based on alkyne or nitrile tags hold promise for highly multiplexed imaging. However, sensing of enzyme activities with Raman probes is difficult because few mechanisms are available to modulate the vibrational response. Here we present a general strategy to prepare activatable Raman probes that show enhanced Raman signals due to electronic preresonance (EPR) upon reaction with enzymes under physiological conditions. We identified a xanthene derivative bearing a nitrile group at position 9 (9CN-JCP) as a suitable scaffold dye, and synthesized four types of activatable Raman probes, which are targeted to different enzymes (three aminopeptidases and a glycosidase) and tuned to different vibrational frequencies by isotope editing of the nitrile group. We validated the activation of the Raman signals of these probes by the target enzymes and succeeded in simultaneous imaging of the four enzyme activities in live cells. Different cell lines showed different patterns of these enzyme activities.

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Summary Observing multiple molecular species simultaneously with high spatiotemporal resolution is crucial for comprehensive understanding of complex, dynamic, and heterogeneous biological systems. The recently reported super-multiplex optical imaging breaks the “color barrier” of fluorescence to achieve multiplexing number over six in living systems, while its temporal resolution is limited to several minutes mainly by slow color tuning. Herein, we report integrated stimulated Raman and fluorescence microscopy with simultaneous multimodal color tunability at high speed, enabling super-multiplex imaging covering diverse molecular contrasts with temporal resolution of seconds. We highlight this technique by demonstrating super-multiplex time-lapse imaging and image-based cytometry of live cells to investigate the dynamics and cellular heterogeneity of eight intracellular components simultaneously. Our technique provides a powerful tool to elucidate spatiotemporal organization and interactions in biological systems.