Mapping Labile Fe2+ and Fe3+ in Living Arabidopsis thaliana Roots: Imaging and Quantification.

Iron (Fe) is involved in numerous key physiological processes due to its transfer or gain electron capacity. However, in excess, Fe is toxic to cells. The understanding of mechanisms governing its homeostasis requires imaging techniques capable of localizing and quantifying Fe and its redox state in situ. The Perls-DAB histochemical staining is a widely used method for visualizing Fe at a cellular level. Other advanced approaches, such as micro X-ray fluorescence and X-ray absorption near-edge structure spectroscopy, also provide information on the spatial distribution and oxidation state of Fe. However, all of these methods require sample fixation before observing Fe localization, which can introduce biases in the results. Recent development in synthetic fluorescent probes now enables the real-time visualization of ferrous (Fe2+) and ferric (Fe3+) forms in living roots, offering a robust and user-friendly approach to study Fe redox distribution in planta. This live imaging approach provides access to Fe dynamics and enables semi-quantitative analyses, thereby opening new avenues for Fe homeostasis research. This chapter provides detailed protocols for applying these methods in Arabidopsis roots.