铁是生命的必需元素，因为它能够参与多种氧化还原反应。然而，铁依赖性氧化还原循环的失调也会产生氧化应激，促进细胞生长、增殖和死亡途径，从而导致衰老、癌症、神经退行性变和代谢疾病。选择性监测松散结合的 Fe(II) 离子（称为不稳定铁池）的荧光探针是研究这种金属营养物的潜在强大工具。然而，这些生物可利用金属库的动态时空性质和强大的荧光猝灭能力给它们的检测带来了挑战。在这里，我们报告了一种基于串联活性的传感和标记策略，该策略能够通过增强细胞保留来对活细胞中的不稳定铁池进行成像。铁绿-1 氟甲基 (IG1-FM) 使用内过氧化物触发器选择性地与 Fe(II) 反应，释放醌甲基化物染料，随后附着到近端生物亲核试剂上，在不稳定铁含量升高的位点提供永久荧光染色。 IG1-FM 成像显示，主要铁储存蛋白铁蛋白通过铁蛋白自噬的降解扩大了不稳定铁库，而核因子红细胞 2 相关因子 2 (NRF2) 抗氧化反应元件 (ARE) 的激活则耗尽了不稳定铁库。我们进一步表明，当用铁螯合剂处理时，NRF2 激活增强且基础不稳定铁降低的肺癌细胞的活力降低。通过将不稳定铁库和 NRF2-ARE 活性与癌症中可药物金属依赖性脆弱性联系起来，这项工作为更广泛地研究过渡金属和抗氧化剂信号通路在健康和疾病中的作用提供了一个起点。

Iron is an essential element for life owing to its ability to participate in a diverse array of oxidation-reduction reactions. However, misregulation of iron-dependent redox cycling can also produce oxidative stress, contributing to cell growth, proliferation, and death pathways underlying aging, cancer, neurodegeneration, and metabolic diseases. Fluorescent probes that selectively monitor loosely bound Fe(II) ions, termed the labile iron pool, are potentially powerful tools for studies of this metal nutrient; however, the dynamic spatiotemporal nature and potent fluorescence quenching capacity of these bioavailable metal stores pose challenges for their detection. Here, we report a tandem activity-based sensing and labeling strategy that enables imaging of labile iron pools in live cells through enhancement in cellular retention. Iron green-1 fluoromethyl (IG1-FM) reacts selectively with Fe(II) using an endoperoxide trigger to release a quinone methide dye for subsequent attachment to proximal biological nucleophiles, providing a permanent fluorescent stain at sites of elevated labile iron. IG1-FM imaging reveals that degradation of the major iron storage protein ferritin through ferritinophagy expands the labile iron pool, while activation of nuclear factor-erythroid 2-related factor 2 (NRF2) antioxidant response elements (AREs) depletes it. We further show that lung cancer cells with heightened NRF2 activation, and thus lower basal labile iron, have reduced viability when treated with an iron chelator. By connecting labile iron pools and NRF2-ARE activity to a druggable metal-dependent vulnerability in cancer, this work provides a starting point for broader investigations into the roles of transition metal and antioxidant signaling pathways in health and disease.