Simultaneous Dual‐Color Stimulated Raman Scattering Biomolecular Imaging with Tailored Supercontinuum Generation and Phase‐Sensitive Detection

We report on the development of a unique dual-color SRS (DC-SRS) microscopy with supercontinuum generation and phase-sensitive detection for simultaneous Raman imaging of vibrational bonds of tissue and cells in both high-wavenumber (2800-3200 cm^-1) and cell-silent regions (1800-2800 cm^-1). With the fine tuning of tailored supercontinuum generation of an 823 nm zero-dispersion photonic crystal fiber, we can obtain intense laser wavelength emissions (700 to 900 nm), serving as two separate pump beams (e.g., 800 and 850 nm) in combination with Stokes beam (1040 nm) for DC-SRS imaging. We introduced the 1/2pi phase-shifted scheme on the Stoke beam to achieve the simultaneous DC-SRS imaging under a single photodiode detection with a 2-channel lock-in amplifier. We have proved the utility of the DC-SRS technique for simultaneous SRS imaging of both C-H (2935 cm^-1) and C-D/C≡C (2110/2216 cm^-1) Raman bands, revealing subcellular metabolic dynamics and precise localization of small alkyne-tagged/deuterated compounds in single living cells. Further, we have also demonstrated the ability of DC-SRS for rapidly quantitative monitoring of cellular metabolism and drug-cell interactions in live tumor spheroid models, suggesting the potential of the simultaneous DC-SRS technique for advancing high-resolution metabolic functional imaging in biological and biomedical systems.