Large-region spectral focusing CARS and TPEF imaging of brain tissues with a low aberration mosaic method.

The multimodal nonlinear optical imaging offers simultaneous multi-component analysis for biological tissues. In this work, large-region multimodal nonlinear optical imaging with a low aberration mosaic method is demonstrated. A multimodal nonlinear optical imaging platform capable of simultaneous two-photon excitation fluorescence (TPEF) and spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) imaging is built. The low aberration mosaic method is proposed and applied for large-region imaging. Through splicing the appropriate area in every TPEF or SF-CARS frame, signal intensity dips at the edge of every frame, caused by aberrations at large scanning angles, are avoided. The effect of this method is verified with TPEF imaging of the pancreatic tissue, and large-region imaging with uniform intensity is achieved. The unlabeled brain tissue of the adult rabbit is chosen as the sample for simultaneous TPEF and SF-CARS imaging. For the multimodal imaging of the brain tissue, the distribution of the fluorescent component, mainly flavin adenine dinucleotide (FAD), and the lipid component in the brain tissue is mapped with TPEF imaging and SF-CARS imaging, respectively. With the relative distribution of FAD and lipids, the morphologies of brain tissue are observed. The polygon area with a weak CARS signal and a strong TPEF signal, an area short of lipid but rich in FAD, is disclosed. Besides, the multimodal nonlinear optical imaging is extended to simultaneous TPEF and SHG imaging by switching the CARS signal detection window to the SHG signal detection window. The relative distribution of FAD and collagen around the vascular structures in the pancreatic tissue was observed.