Quantitative three-dimensional absorption imaging of pigments and stains with brightfield microscopy

Optical absorption is a primary, label-defining contrast across biology, pathology, and materials science, yet three-dimensional quantitative absorption imaging has remained largely inaccessible to the brightfield microscopes used in everyday practice. We introduce quantitative absorption tomography (QAT), which recovers volumetric distributions of the extinction coefficient by treating brightfield image formation as a linear inverse problem in logarithmic intensity space and inverting a three-dimensional absorption optical transfer function. Under weak-scattering conditions, QAT yields spectrally resolved, three-dimensional absorption maps from through-focus image stacks acquired on standard brightfield platforms, without interferometry, coherent illumination, or sample rotation. We use QAT to track melanin dynamics in living melanoma cells without exogenous labels, image pigment organization in intact Petunia hybrida petals in vivo, and reconstruct chromogenic contrast across large H&E-stained human tissue volumes. By establishing absorption as a directly measurable volumetric quantity within standard brightfield workflows, QAT positions chromogenic contrast as a quantitative axis alongside fluorescence- and refractive-index-based imaging.