Frequency Modulation Stimulated Raman Scattering Microscopy through Polarization Encoding

Stimulated Raman scattering (SRS) microscopy is a powerful method for imaging molecular distributions based on their intrinsic vibrational contrast. However, despite a growing list of biological applications, SRS is frequently hindered by a parasitic background signal which both overpowers the signal in low-signal applications and makes the extraction of quantitative information from images challenging. Frequency modulation (FM) has been used to suppress this parasitic background. However, many FM-SRS methods require either the acquisition of multiple images or the addition of multiple optomechanical components and an extensive realignment procedure. Herein, we report a new procedure for alignment-free FM-SRS utilizing polarization encoding. We demonstrate the efficacy of this approach, along with parabolic amplification of the Stokes pulse, at removing parasitic background signals in SRS microscopy applications. We further highlight how this technique can be used to suppress Raman signals from major molecular species to unveil spectral signatures from nucleic acids in both murine brain tissue and whole blood. Due to its ease of use and demonstrated experimental capabilities, we expect this technique to see broad use in the SRS microscopy community.