Dual-phase stimulated Raman scattering microscopy for real-time two-color imaging

Label-free histology with chemical contrast has great potential for rapid intraoperative diagnosis. Two-color stimulated Raman scattering (SRS) microscopy has shown success in label-free digital histology with diagnostic results similar to those of hematoxylin and eosin stain. However, achieving real-time two-color SRS imaging has been a challenge. We have precisely engineered the pulse profiles of Stokes beams, and fully utilized in-phase (X) and quadrature (Y) outputs of a phase-sensitive lock-in amplifier to realize simultaneous two-color imaging. Such a method could reach the maximum speed as in single-color SRS, and has proven its robustness and advantages in real-time histology as well as in vivo imaging of live animals, both in transmission and epi modes. Moreover, this method could be conveniently adapted to other pump–probe-based microscopes.