Monitoring hydrogen polysulfide during ferroptosis with a two-photon fluorescent probe

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.