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Heterogenous zonation plays a crucial role in liver physiology and pathology. Extracellular collagen fiber deposition and intracellular lipid accumulation are hallmarks of many liver diseases, but their zonal distribution and pathological significance remains unknown. Here, we established a label-free multimodal nonlinear optical (NLO) microscopy that integrated stimulated Raman scattering (SRS) imaging and compositional analysis of neutral lipids, second harmonic generation (SHG) imaging of collagen fibers, and two-photon excited fluorescence (TPEF) imaging of auto-fluorescent metabolites to study liver zonation in situ. By analyzing the liver tissues obtained from carbon tetrachloride (CCl4) induced mouse centrilobular fibrosis model, our imaging data revealed that the amount of collagen fibers increased rapidly in central vein (CV) area, while significantly slower in portal vein (PV) area. Along with the collagen fibers deposition, the accumulation of triglycerides (TGs) was also significantly increased in CV area, but almost absent in PV area. Within the total TGs, although the amount of unsaturated TGs increased significantly in CV area, the lipid unsaturation degree, defined as the number of C=C bands on each fatty acid chain, significantly decreased in the late-stage compared to the early-stage fibrosis, which was likely due to the change of stearoyl-coenzyme A desaturase-1 (SCD1) expression. Meanwhile, lipofuscin and malondialdehyde, products of lipid peroxidation, were found to be gradually accumulated in CV area as fibrosis progressed. Interestingly, the heterogeneous distribution of collagen fibers and lipids were also found in fibrotic livers in patients. Our study by label-free multimodal NLO microscopy discovered the liver zonation of TG and collagen fiber, which could serve as valuable biomarkers for assessment of liver fibrosis progression.
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