Characterization of lipid composition and localization at different stages of nonalcoholic fatty liver disease using stimulated Raman scattering microscopy

Abstract

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid in the hepatocytes (steatosis), which can progress to fibrosing steatohepatitis (NASH). The molecular mechanisms driving this progression are insufficiently understood. A leading hypothesis is lipotoxicity, which postulates that specific lipid species can trigger the cascade of inflammation leading to liver damage. In this study, we used label-free stimulated Raman scattering (SRS) microscopy to characterize the distribution of free cholesterol, cholesteryl esters, and triglycerides of different saturation levels in liver tissue from patients with histologically diagnosed NAFLD versus NASH. By probing the intrinsic vibrational frequencies of lipid molecules in the C–H and the fingerprint regions, we can localize and classify different lipid molecules in lipid droplets via spectral unmixing. We report our developed image acquisition and processing pipeline in this paper and demonstrate example applications such as examining the composition of previously described cholesterol crystals. We discovered that most of the birefringent liquid crystals presumed to be free cholesterol crystals in NASH tissues are predominantly composed of saturated cholesteryl esters. Our method provides a detailed characterization of the lipid composition in NAFLD tissues and allows us to probe further into the potential mechanism of NASH development.