摘要：SummaryObserving 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.Graphical abstractDisplay OmittedHighlights•Integrated SRS and fluorescence microscopy with fast tunability has been developed•Eight-color live-cell imaging can be conducted with a temporal resolution of seconds•Super-multiplex time-lapse imaging reveals complex organelle interactions•Super-multiplex image-based cytometry accesses high-dimensional heterogeneityOptics; Cell biology; Biological sciences research methodologies
