Managing Autofluorescence in Spectral Flow Cytometry for Macrophage Identification in the Liver

Autofluorescence (AF) in biological tissues arises from the natural emission of light by intra- and extracellular molecules upon light absorption. Conventional flow cytometry cannot correct for cellular AF, leading to distorted signals and measurement errors. While spectral flow cytometry enables AF visualization and extraction, accurately correcting for AF remains challenging in complex biological samples containing multiple cell types with distinct AF properties, such as the liver. Additionally, pathological processes such as inflammation and fibrosis can alter tissue composition and activate specific cell types, further modifying AF characteristics across experimental conditions. Macrophages are among the most autofluorescent immune cells, exhibiting fluorescence emission across the entire spectrum of light. Recent studies have demonstrated substantial heterogeneity in the phenotypes of resident and recruited macrophages both in the healthy liver and during Metabolic Dysfunction-Associated Steatohepatitis (MASH). Given their critical role in liver disease pathophysiology, we developed a spectral flow cytometry approach to identify and analyze all macrophage subpopulations in healthy and MASH murine livers. Our findings show that healthy, steatotic and MASH livers exhibit distinct and heterogeneous AF signatures. Furthermore, inadequate AF extraction compromised accurate quantification of hepatic macrophages and differentiation of macrophage subsets.