Polychromatic Adaptive Optics‐Assisted Stimulated Raman Scattering Microscopy Enables Deep Tissue Chemical Imaging

Stimulated Raman scattering (SRS) microscopy has revolutionized label‐free chemical imaging by leveraging molecular vibrations to reveal biochemical compositions of cells and tissues. However, the wavelength‐dependent scattering in tissues distorts laser beam wavefronts, significantly degrading image quality and depth in SRS imaging. Here, the study introduces a unique polychromatic adaptive optics‐assisted SRS (PAO‐SRS) microscopy, enabling high‐sensitivity, high‐resolution, deep tissue 3D imaging in turbid samples. By employing a spatial light modulator (SLM) to independently correct aberrations in incident SRS beams, PAO‐SRS effectively addresses tissue‐induced, wavelength‐dependent scattering, thereby maintaining a high signal‐to‐noise ratio in deep tissue regions. PAO‐SRS provides remarkable ∼3‐fold enhancement in SRS intensity in deep brain tissue, leading to a 3‐fold increase in SRS imaging depth. Further, PAO‐SRS enables mechanical z‐scanning‐free, aberration‐free 3D chemical imaging by electronically controlling the axial positions of laser foci associated with the corresponding aberration corrections at each tissue depth. An enhanced imaging resolution and intensity in zebrafish liver region is demonstrated using PAO‐SRS, and also visualizes global metabolic variations in live zebrafish liver, unraveling distinct chemical differences between normal and tumor liver under starvations. PAO‐SRS holds great potential for advancing label‐free, high‐resolution, 3D chemical imaging in deep tissue, opening new avenues in biomedical research and diagnostics.