For its label-free imaging capability with chemical specificity, Raman microscopy has found various applications in biology, medicine, pharmacology, and material science. In imaging of moving samples and/or a large number of objects, however, spontaneous Raman imaging falls short due to its slow signal acquisition time. Multicolor stimulated Raman scattering (SRS) microscopy is a promising approach to this end because it can probe the vibrational signatures of molecules at multiple vibrational frequencies with orders of magnitude higher sensitivity than spontaneous Raman scattering. Here we report our two recent achievements that boost the chemical imaging speed of multicolor SRS microscopy. The first one is multicolor SRS imaging that operates at 110 frames per second by frame-by-frame wavenumber tuning. Using this method, we demonstrated video-rate single-cell imaging of intracellular metabolites in live Euglena gracilis, a highly motile microalgal species. We revealed large cell-to-cell variations in the amounts of metabolites, which is effective for efficient biomaterial engineering. The second one is four-color SRS imaging that runs at a pixel dwell time of 0.2 µs per pixel, enabled by fast wavelength switching of laser pulses. We used it to demonstrate SRS-based chemical imaging of polymer beads and living cells. Our SRS system’s fast imaging capability made high-throughput Raman imaging possible as well as blur-free Raman imaging, which are highly useful in diverse applications including cell screening and intraoperative diagnostics.

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Yuta Suzuki, Keisuke Goda, and Yasuyuki Ozeki, "High-speed multicolor SRS imaging (Conference Presentation)," Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104981M (Presented at SPIE BiOS: January 30, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2291213.5751631610001.