Whole-organism 3D Quantitative Characterization of Zebrafish Melanin by Silver Deposition Micro-CT

Melanin-rich zebrafish melanophores are used to study pigment development, human skin color, and as a large-scale screening phenotype. To facilitate more detailed whole-body, computational analyses of melanin content and morphology, we have combined X-ray microtomography (micro-CT), a non-destructive, full-volume imaging modality, with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, for both dramatic and subtle phenotypes not previously described. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, three-dimensional computational phenomic analysis of a particular cell type at cellular resolution, with potential applications in other model organisms and human melanoma biopsies. Whole-organism, high-resolution phenotyping is a challenging ideal, but provides superior context for functional studies of mutations, diseases, and environmental influences.