Super resolution microscopy-based assessment of organellar redox-active iron transfer and its relevance for...

Intracellular iron is essential for numerous biological processes, yet its redox activity makes it potentially cytotoxic. Because of this, a tight regulation of its cellular compartmentalization is required. Lysosomes and mitochondria play central roles in iron metabolism. Lysosomes are crucial for iron redistribution after its endocytosis, while mitochondria utilize it for heme and Fe-S cluster synthesis. Disruption of the functional crosstalk between these two organelles can lead to iron dyshomeostasis and ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation. Recent evidence highlights the importance of mitochondria-lysosome contact sites (MLCs) in mediating iron trafficking, particularly under pathological conditions. However, studying these nanoscopic, dynamic structures poses significant technical challenges. Here, we describe a novel live-cell imaging protocol combining super-resolution structured illumination microscopy (SIM) with organelle-specific dyes and a selective mitochondrial Fe(II) probe to visualize MLC formation and track iron transfer in real time. This approach enables the precise investigation of subcellular iron dynamics and their implications for ferroptosis and disease.