Label-Free Nanoimaging of Neuromelanin in the Brain by Soft X-ray Spectromicroscopy

A hallmark of Parkinson's disease is the death of neuromelanin-pigmented neurons, but the role of neuromelanin is unclear. The in situ characterization of neuromelanin remains dependent on detectable pigmentation, rather than direct quantification of neuromelanin. We show that direct, label-free nanoscale visualization of neuromelanin and associated metal ions in human brain tissue can be achieved using synchrotron scanning transmission x-ray microscopy (STXM), through a characteristic feature in the neuromelanin x-ray absorption spectrum at 287.4 eV that is also present in iron-free and iron-laden synthetic neuromelanin. This is confirmed in consecutive brain sections by correlating STXM neuromelanin imaging with silver nitrate-stained neuromelanin. Analysis suggests that the 1s-sigma* (C-S) transition in benzothiazine groups accounts for this feature. This method illustrates the wider potential of STXM as a label-free spectromicroscopy technique applicable to both organic and inorganic materials.