Peroxynitrite (ONOO−) is a highly reactive and destructive oxygen species that is formed by the diffusion-controlled reaction of nitric oxide (NO) with superoxide anion radical (O2•-) [1]. Known for its potent reactivity in the reactive oxygen species (ROS) family, ONOO− readily induces irreversible damage to lipids, proteins, and DNA, contributing significantly to cell death. High ONOO− levels have been implicated in the pathological processes of various oxidative stress-related diseases, such as ischemia-reperfusion (IR) injury, inflammation, and neurodegeneration [[2], [3], [4]]. Extensive experimental evidence has highlighted the significant benefits of therapeutic strategies aiming at developing peroxynitrite scavengers and peroxynitrite decomposition catalysts for modulating its formation and degradation [5]. Therefore, comprehensively monitoring ONOO− at the whole body and tissue level would greatly help understand its precise roles in various diseases.

However, it isn't easy to accurately detect and visualize the production of ONOO− in biological systems due to its high reactivity and very short lifespan. Although several efforts have been undertaken over the past decade to determine the production of ONOO−, the combination of fluorescence and mass spectrometry imaging (MSI) is rarely reported. A significant advantage of combining multiple molecular imaging modalities, as opposed to relying on a single modality, is the ability to provide comprehensive information that represents multiple perspectives. Fluorescence imaging provides a live imaging approach for monitoring disease onset and progression in organisms [[6], [7], [8]], meanwhile, MSI allows for mapping the spatial distribution of individual metabolites in tissues with high selectivity and sensitivity, particularly for identifying characteristic molecules that reflect the fine-scale metabolic heterogeneity of tissues [9].

A major challenge in the integration of the strengths of two imaging modalities is the development of one probe to meet the specificity and applicability of each imaging technique. Therefore, a novel tetrazine-hemicyanine-based probe (TZN-HCY) was rationally synthesized which displayed high selectivity and sensitivity toward ONOO− and was compatible with dual-modal imaging: fluorescence and matrix-assisted laser desorption/ionization (MALDI) MS imaging. The design of the probe capitalized on the unique properties of 3,6-disubstituted tetrazine, which served as an ONOO−-responsive moiety. Additionally, the inherent positive charge of the hemicyanine scaffold enhanced detection sensitivity in MALDI MSI. Due to its high selectivity, sensitivity, and versatility, in vivo measurements of ONOO− change were performed on the livers of hepatic ischemia-reperfusion injury (HIRI) model mice using fluorescence imaging, and the tissue-specific distribution of ONOO− in the microregions of liver sections was successfully visualized using MALDI MSI. We envision that the dual-model imaging probe TZN-HCY could provide a useful tool for tracking ONOO− in various pathophysiological environments.