Super-multiplex imaging of cellular dynamics and heterogeneity by integrated stimulated Raman and fluorescence microscopy
Jingwen Shou,
Robert Oda,
Fanghao Hu,
Keiko Karasawa,
Mutsuo Nuriya,
Masato Yasui,
Bruce Shiramizu,
Wei Min,
Yasuyuki Ozeki
Affiliations
Jingwen Shou
Department of Electrical Engineering and Information Systems, The University of Tokyo, Tokyo 113-8656, Japan
Robert Oda
Department of Electrical Engineering and Information Systems, The University of Tokyo, Tokyo 113-8656, Japan; Department of Molecular Biosciences and Bioengineering, The University of Hawaii, Manoa, 1955 East West Road, Honolulu, Hawaii 96822, USA; Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, 651 Ilalo Street, Honolulu, Hawaii 96813, USA; Department of Pharmacology School of Medicine, Keio University, Tokyo 160-8582, Japan
Fanghao Hu
Department of Chemistry, Columbia University, New York, New York 10027, USA; Department of Chemistry, Tsinghua University, Beijing 100084, China; Corresponding author
Keiko Karasawa
Department of Pharmacology School of Medicine, Keio University, Tokyo 160-8582, Japan
Mutsuo Nuriya
Department of Pharmacology School of Medicine, Keio University, Tokyo 160-8582, Japan
Masato Yasui
Department of Pharmacology School of Medicine, Keio University, Tokyo 160-8582, Japan
Bruce Shiramizu
Department of Molecular Biosciences and Bioengineering, The University of Hawaii, Manoa, 1955 East West Road, Honolulu, Hawaii 96822, USA; Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, 651 Ilalo Street, Honolulu, Hawaii 96813, USA
Wei Min
Department of Chemistry, Columbia University, New York, New York 10027, USA; Kavli Institute for Brain Science, Columbia University, New York, New York 10027, USA; Corresponding author
Yasuyuki Ozeki
Department of Electrical Engineering and Information Systems, The University of Tokyo, Tokyo 113-8656, Japan; Corresponding author
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.
