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

Abstract

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.

Super-resolution vibrational imaging based on photoswitchable Raman probe

“…The epi-fluorescence is spatially filtered by a confocal pinhole and spectrally filtered to remove laser light. Then the fluorescence is introduced into a tunable optical bandpass filter and finally detected by a photomultiplier to generate the fluorescence signal 8 .…”

“…Furthermore, techniques of multicolor super-resolution fluorescence imaging have been developed 3–4 , though it is difficult to greatly increase the number of resolvable colors due to the large bandwidth of the fluorescence signals 5 . In contrast, Raman scattering, which probes molecular vibrational states, can generate sharp spectral signatures with about 50 times narrower linewidth than that of fluorescence 6–8 . Hence, extending Raman microscopy beyond the diffraction limit seems a promising approach to increase the degree of multiplexing for far-field super-resolution light microscopy.…”

Miniaturized Chemical Tags for Optical Imaging

“…Through integration of fluorescence microscopy into a similar SRS platform, Ozeki and co-workers demonstrated faster visualization of multiplexed samples using an image-based cytometry approach. [94] An interesting alternative design for multiplexed miniaturized probes was recently reported via coupling of Ramanactive alkyne and nitrile moieties to NIR chromophores. [90] By matching the molecular absorption of the xanthene chromophore to be close to -yet not matching the SRS excitation wavelength-the authors achieved a dramatic enhancement of the Raman scattering cross-section (e.g., up to 10 5 -fold).…”

“…In this study, the conjugation of Rdots to antibodies and aptamers was used to analyze cell surface protein abundance, endocytosis activity and metabolic dynamics. Through integration of fluorescence microscopy into a similar SRS platform, Ozeki and co‐workers demonstrated faster visualization of multiplexed samples using an image‐based cytometry approach [94] …”

Probing Methionine Uptake in Live Cells by Deuterium Labeling and Stimulated Raman Scattering

“…Another advantage of our multicolor SRS imaging system is its flexible ability in the allocation of multiple spectral points. This work extends the applicability of SRS imaging with deuterium labeling beyond the previously seen abundant amino acids, 24,37 glucose, 38,39 choline, 40 and water 41 and together with these previous works paves the way for multiplex SRS imaging 29 of a whole range of amino acids including less-abundant species and other biomolecules and potentially their metabolites.…”