Near-resonance enhanced label-free stimulated Raman scattering microscopy with spatial resolution near 130 nm

High-resolution optical microscopes that can break 180 nm in spatial resolution set to conventional microscopies are much-needed tools. However, current optical microscopes have to rely on exogenous fluorescent labels to achieve high resolution in biological imaging. Herein, we report near-resonance enhanced label-free stimulated Raman scattering (SRS) microscopy with a lateral resolution near 130 nm, in which the high-resolution image contrast originates directly from a low concentration of endogenous biomolecules, with sensitivity gains of approximately 23 times. Moreover, by using a 0.3-m-long optical fiber, we developed hyperspectral SRS microscopy based on spectral focusing technology. Attributed to enhancements in spatial resolution and sensitivity, we demonstrated high-resolution imaging of three-dimensional structures in single cells and high-resolution mapping of large-scale intact mouse brain tissues in situ. By using enhanced high-resolution hyperspectral SRS, we chemically observed sphingomyelin distributed in the myelin sheath that insulates single axons. Our concept opens the door to biomedical imaging with ~130 nm resolution.