Quantitative profiling of lysosomal accumulation through label-free biomarkers via High-Content Holo-Tomographic Flow Cytometry

Lysosomal storage diseases (LSDs) are genetic disorders caused by enzyme deficiencies that lead to lysosomal dysfunction and progressive cell damage. Accurate visualization and quantification of lysosomes are essential for understanding disease progression and developing effective therapies. Here, for the first time, we successfully identified and characterized lysosomes using an innovative Holo-Tomographic Flow Cytometry (HTFC) technique, which allows label-free, high-content, and high-throughput 3D imaging of lysosomal compartments in single live cells. This breakthrough could revolutionize traditional gold-standard methods overcoming the actual limitations. Leveraging this technology, we discovered novel biomarkers of lysosomal accumulation in LSD-affected cells. In fact, by generating refractive index tomograms, we achieved accurate measurement and comprehensive 3D visualization of cytoplasmic lysosomal aggregation in suspended single cells. Through experimental validation and advanced computational analyses, we identified a quantitative correlation between the 3D lysosomal architecture and the efficacy of various therapeutic strategies, including genetic and pharmacological interventions. This work represents a significant advance in lysosomal research, paving the way for improved diagnostics and the development of targeted therapies for LSDs.