In recent studies, stimulated Raman scattering (SRS) and transient absorption microscopy (TAM) have been employed for label-free mapping of biomolecules (e.g., proteins and lipids) in brain tissues and hemoglobin in red blood cells, respectively. In this study, we combined SRS and TAM to simultaneously image cell densities and capillary structure in vivo at the highest reported imaging depth, ~300 µm, for either technique. This multimodal approach resulted in label-free identification of endothelial cells and pericytes in vivo with 90% accuracy using a machine learning classifier. Simultaneous two-photon excited fluorescence microscopy serving as the ground truth.
Stimulated Raman Scattering (SRS) microscopy is a powerful method for imaging molecular distributions based on their intrinsic vibrational contrast. However, SRS is hindered by a parasitic background signal which often overpowers the signal in low-signal applications. Frequency modulation (FM) has been used to suppress this parasitic background. However, many FM-SRS methods require the addition of multiple optomechanical components and extensive realignment. We report a new approach for alignment-free FM-SRS. In conjunction with a parabolically amplified Stokes pulse, we demonstrate near complete background suppression and also utilize the technique to increase the contrast of minority species in heterogeneous biological samples.
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