XANES Spectroscopic Analysis of Frozen-Hydrated Brain Tissue Using a N2 Cryo-Stream Best Preserves In Vivo Fe and Cu Speciation.

Redox-active transition metal ions, such as Fe and Cu, are critical to support healthy brain function. Unfortunately, Fe and Cu homeostasis is a double-edged sword in the brain, where disturbed amounts or changes in their distribution or chemical form can drive oxidative stress and tissue damage. Characterising the chemical form of Fe and Cu in situ in brain tissue has historically been difficult because few analytical methods are sensitive to the different oxidation states and bonding environments in which Fe and Cu can exist. Recently, XANES spectroscopy has become a popular method for studying Fe and Cu speciation in situ in biological systems. However, one unanswered question in using XANES spectroscopy to study Fe and Cu speciation in situ is how sample preparation affects metal oxidation state and coordination environment. This investigation has therefore compared XANES spectra collected from air-dried brain tissue with spectra from frozen-hydrated brain tissue. The results highlight that to prevent artificial sample-preparation-induced oxidation and detect Fe and Cu as close as possible to in vivo conditions, analysis should be performed on frozen-hydrated tissues.