SignificanceThe molecular mechanisms driving the progression from nonalcoholic fatty liver (NAFL) to fibrosing steatohepatitis (NASH) are insufficiently understood. Techniques enabling the characterization of different lipid species with both chemical and spatial information can provide valuable insights into their contributions to the disease progression.AimWe extend the utility of stimulated Raman scattering (SRS) microscopy to characterize and quantify lipid species in liver tissue sections from patients with NAFL and NASH.ApproachWe applied a dual-band hyperspectral SRS microscopy system for imaging tissue sections in both the C–H stretching and fingerprint regions. The same sections were imaged with polarization microscopy for detecting birefringent liquid crystals in the tissues.ResultsOur imaging and analysis pipeline provides accurate classification and quantification of free cholesterol, saturated cholesteryl esters (CEs), unsaturated CE, and triglycerides in liver tissue sections. The subcellular resolution enables investigations of the heterogeneous distribution of saturated CE, which has been under-examined in previous studies. We also discovered that the birefringent crystals, previously found to be associated with NASH development, are predominantly composed of saturated CE.ConclusionsOur method allows for a detailed characterization of lipid composition in human liver tissues and enables further investigation into the potential mechanism of NASH progression.
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.
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