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Summary
Melanin within melanosomes exists as eumelanin or pheomelanin. Distributions of these melanins have been studied extensively within tissues, but less often within individual melanosomes. Here, we apply X-ray fluorescence analysis with synchrotron radiation to survey the nanoscale distribution of metals within purified melanosomes of mice. The study allows a discovery-based characterization of melanosomal metals, and, because Cu is specifically associated with eumelanin, a hypothesis-based test of the “casing model” predicting that melanosomes contain a pheomelanin core surrounded by a eumelanin shell. Analysis of Cu, Ca, and Zn shows variable concentrations and distributions, with Ca/Zn highly correlated, and at least three discrete patterns for the distribution of Cu vs. Ca/Zn in different melanosomes – including one with a Cu-rich shell surrounding a Ca/Zn-rich core. Thus, the results support predictions of the casing model, but also suggest that in at least some tissues and genetic contexts, other arrangements of melanin may co-exist.
Full-field transmission hard X-ray microscopy (TXM) has been widely applied to study morphology and structures with high spatial precision and to dynamic processes. Zernike phase contrast (ZPC) in hard X-ray TXM is often utilized to get an in-line phase contrast enhancement for weak-absorbing materials with little contrast differences. Here, following forward image formation, we derive and simplify the contrast transfer functions (CTFs) of the Zernike phase imaging system in TXM based on a linear space-shift-invariant imaging mode under certain approximations. The CTFs in ZPC in their simplified forms show a high similarity to the one in free-space propagation X-ray imaging systems.
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