QUILPEN decouples pigment absorption and organelle scatter in live melanocytic cells

Pigmentation is a defining feature of melanocytic cells, and melanogenesis, the biosynthesis and compartmentalization of pigment into lysosome-like melanosomes, is tightly linked to cell state and developmental programs. However, the optical complexity of melanin-rich organelles complicates efforts to study melanogenesis in live cells. Traditional optical measurements conflate pigment absorption with light scattering from intracellular granularity, limiting biological insight. Our pipeline, QUILPEN (QUantitative Imaging of Label-free Pigment-associated ENtities), uses a custom LED-array microscope to independently quantify transmitted, scattered, and absorbed light in live, unlabeled melanocytic cells. QUILPEN builds off our labs prior work in LED-based DPC and Quadrant Dark Field, or QDF, which reduce scatterrelated edge-effects. Our imaging workflow produces temporally resolved, multi-channel images to characterize the biophysical signature at single-cell resolution. Using melanoma cell lines with diverse pigmentation states, we show that acral melanoma cells display tightly correlated scatter and absorption signals, consistent with pigment-laden melanosomes driving both granularity and light absorption. By analyzing well-characterized melanoma models and perturbing pigment synthesis, we define how QUILPEN signals relate to melanin production, melanosome abundance, and general organelle content. Because QUILPEN enables longitudinal tracking without labels, it allows direct measurement of melanosome dynamics within individual cells and lineages. By separately quantifying absorption and scatter, QUILPEN reveals the sources of optical heterogeneity in melanocytic cells and links optical signatures to underlying cell physiology.