QUILPEN provides independent and label-free single-cell quantification of pigmentation dynamics and organelle content.

The relationship between melanogenesis, pigmentation, and melanocyte behavior is complex. In melanocytes, pigmentation is often associated with differentiation, yet mature melanocytes vary in pigment content. In melanoma, pigmentation-linked transcriptional programs may have prognostic value, but visual assessments of tumor pigmentation have yielded inconsistent results. Progress linking pigmentation phenotypes to cell state has been limited by a lack of tools that can directly and dynamically quantify melanin content in live cells. Here we present QUantitative Imaging of Label-free Pigment-associated ENtities (QUILPEN), a label-free multi-modal imaging technique that combines quantitative phase imaging (QPI), quadrant darkfield (QDF), and absorption imaging, to independently capture light that has been transmitted, scattered, and absorbed. This non-destructive method enables live-cell imaging over multiple days without labels. We show absorption as a reliable readout of melanin content, which can be decoupled from melanosome content detected by QDF, which measures scattered light. Applying QUILPEN to melanoma cells before and during repigmentation, we find that melanin content is highly heterogeneous, and that this heterogeneity is reinstated upon repigmentation. Lineage tracking further reveals that melanin synthesis rates are heritable and can be transmitted both symmetrically and asymmetrically. QUILPEN enables real-time quantification of pigmentation dynamics and cell-level heterogeneity. Significance: Pigmentation is a defining feature of melanocytic cells and linked to behavior, yet melanin's optical properties complicate direct measurement with existing approaches. Taking advantage of the absorbing nature of pigment and scattering properties of organelles, we can independently track pigment and organelle content over time and along cell lineages in a label-free manner to reveal population heterogeneity and patterns of inheritance. Research Highlights: QUILPEN is a label-free, live cell imaging pipeline that independently quantifies pigment and organelles in melanocytic cells. Tracking pigment and organelle content along cell lineages reveals varied inheritance of pigment-associated features.