Ir-ImNO is a mitochondria-targeting two-photon probe with excellent cell membrane permeability, capable of detecting both endogenous and exogenous nitric oxide (NO). It allows monitoring of different immune response states in macrophages through a variety of imaging techniques, including single-photon and two-photon phosphorescence imaging as well as phosphorescence lifetime imaging. Ir-ImNO can be used in research for the clinical monitoring of immunotherapy.