Beyond In Vitro Brightness: A Matched-Probe Study of Chemiluminescence Contrast for In Vivo Peroxynitrite Imaging

Probe selection for in vivo chemiluminescence imaging commonly relies on intensity and tissue-overlay measurements performed in vitro, yet these measurements do not reproduce the activation background, probe distribution, and optical environment of a living animal. Here, we test how measurement context affects probe ranking using two ONOO--responsive bicyclic dioxetanes, Probe 1 and Probe 2, that share the same arylboronate trigger and closely related core architecture but emit at different wavelengths. In black 96-well plates after ONOO- activation, the visible-emitting Probe 2 produced approximately 350-fold greater photon output than Probe 1, and Probe 2 retained the larger signal-to-background ratio in a chicken-tissue overlay experiment. These in vitro and ex vivo measurements would both favor Probe 2. In living mice, however, the ranking by analytical contrast changed. Probe 1 afforded a subcutaneous signal-to-background ratio of 489 and produced an approximately 7-fold challenged-to-control contrast in an Escherichia coli-induced peritonitis model, compared with approximately 1.5-fold for Probe 2, the absolute signal difference narrowed to approximately 3-fold. This environment-dependent reordering shows that plate intensity and tissue overlays alone can misrank probes intended for in vivo analysis. Emission wavelength, preactivation background, tissue transmission, and model-specific signal distribution must be evaluated together.