Super-multiplex imaging of cellular dynamics and heterogeneity by integrated stimulated Raman and fluorescence microscopy

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 severalminutes 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.

Automated summary

Observing multiple molecular species simultaneously with high spatiotemporal resolution is crucial for comprehensive understanding of biological systems. The super-multiplex optical imaging technique breaks the color barrier of fluorescence, achieving multiplexing number over six in living systems. Integrated stimulated Raman and fluorescence microscopy enables super-multiplex imaging with high speed and temporal resolution of seconds, providing a powerful tool to elucidate spatiotemporal organization and interactions in biological systems.