Simultaneous dual-Raman-shift scanning-free stimulated Raman scattering microscopy for label-free biomolecular imaging.

Conventional stimulated Raman scattering (SRS) microscopy requires time-consuming Raman-shift scanning to achieve chemical specificity, limiting its application in real-time biological studies. We present simultaneous dual-Raman-shift scanning-free SRS (SDRSRS) microscopy, which captures different distinct molecular vibrational signatures simultaneously without spectral scanning, achieving more than 10-fold faster imaging while maintaining high chemical specificity. The system uses birefringent crystals to create controlled optical path differences and orthogonal polarizations, enabling dual-phase modulation with custom dual-channel detection. The collinear balanced detection mode provides 11 dB signal-to-noise ratio improvement, and the system is compatible with other real-time imaging modalities. SDRSRS enables simultaneous CH₃ and CH₂ mapping to reveal lipid droplet dynamics in living cells, quantitatively distinguishes immunotherapy responses in liver cancer models, and provides rapid label-free classification of human colorectal cancer. This technique enables rapid and simultaneous dual vibrational contrast, providing chemically specific imaging for live metabolic monitoring, clinical pathology, and drug-response studies.