Hydrogen polysulfide (H₂S_n, n > 1), a member of reactive sulfur species (RSS), is primarily generated during the crosstalk between H₂S and reactive oxygen species (ROS), which plays important role in physiological and pathological processes. Ferroptosis is a new non-classical mode of cell death, in which ROS-associated lipid peroxidation and iron-dependent accumulation are the main features. However, the biological effects of H₂S_n on ferroptosis and the detailed mechanisms of action remain poorly understood. Thus, there is an urgent need to develop highly selective and sensitive chemical tools for monitoring H₂S_n in living cells. Herein, we develop a two-photon fluorescent probe (PSP) for specifically imaging H₂S_n in live cells and tumor spheroids. This probe exhibited a sensitive and selective response to H₂S_n, which had been used for imaging exogenous and endogenous H₂S_n in living cells by confocal imaging and high content imaging. PSP exhibits excellent photo-stability and two-photon imaging performance when irradiating at 880 nm in 3D HeLa multicellular tumor spheroids. Importantly, our studies revealed that H₂S_n levels were significantly up-regulated during ferroptosis. These excellent properties ensure that PSP is a promising two-photon probe for exploring the biological and pathological effects of H₂S_n during ferroptosis.

多硫化氢（H ₂ S _n，n> 1）是活性硫物质（RSS）的成员，主要是在H ₂ S与活性氧物质（ROS）之间的串扰期间产生的，在生理和病理过程中起重要作用。Ferroptosis是一种新的非经典细胞死亡模式，其中ROS相关脂质过氧化和铁依赖性积累是主要特征。但是，对H ₂ S _n对肥大症的生物学作用和详细的作用机理仍然知之甚少。因此，迫切需要开发高度选择性和灵敏的化学工具来监测H ₂ S _n在活细胞中。本文中，我们开发了一种双光子荧光探针（PSP），用于在活细胞和肿瘤球体中对H ₂ S _n进行专门成像。该探针对H ₂ S _n表现出敏感和选择性的反应，已通过共聚焦成像和高含量成像对H ₂ S _n进行了成像。当在3D HeLa多细胞肿瘤球体中以880 nm照射时，PSP表现出出色的光稳定性和两光子成像性能。重要的是，我们的研究表明H ₂ S _n铁水平在肥大病期间显着上调。这些优异的性能确保PSP是一种有前途的双光子探针，可用于探索肥大病期间H ₂ S _n的生物学和病理学作用。