TauSTED super-resolution imaging of labile iron in primary hippocampal neurons

Iron dyshomeostasis is involved in many neurological disorders, particularly neurodegenerative diseases where iron accumulates in various brain regions. Identifying mechanisms of iron transport in the brain is crucial for understanding the role of iron in healthy and pathological states. In neurons, it has been suggested that iron can be transported by the axon to different brain regions in the form of labile iron; a pool of reactive and exchangeable intracellular iron. Here we report a novel approach to imaging labile ferrous iron, Fe(II), in live primary hippocampal neurons using confocal and TauSTED (stimulated emission depletion) microscopy. TauSTED is based on super-resolution STED nanoscopy, which combines high spatial resolution imaging (<40 nm) with fluorescence lifetime information, thus reducing background noise and improving image quality. We applied TauSTED imaging utilizing biotracker FerroFarRed Fe(II) and found that labile iron was present as submicrometric puncta in dendrites and axons. Some of these iron-rich structures are mobile and move along neuritic pathways, arguing for a labile iron transport mechanism in neurons. This super-resolution imaging approach offers a new perspective for studying the dynamic mechanisms of axonal and dendritic transport of iron at high spatial resolution in living neurons. In addition, this methodology could be transposed to the imaging of other fluorescent metal sensors.