A Palette of Fluorescent Probes with Varying Emission Colors for Imaging Hydrogen Peroxide Signaling in Living Cells

TLDR

The study introduces a family of color‑varying fluorescent probes for selective imaging of physiologically relevant hydrogen peroxide. The probes, based on fluorescein and rhodol scaffolds, employ a boronate‑to‑phenol switch that selectively reacts with H2O2, allowing tunable excitation/emission and sensitivity, and were validated in macrophages and A431 cells and used alongside the APF probe for multicolor imaging. Using the dual‑probe system, the authors distinguished H2O2 from HOCl in live macrophages, identified three phagosome types with distinct ROS profiles, and demonstrated that a palette of colored probes enables simultaneous monitoring of multiple ROS to dissect oxidative biology.

Abstract

We present a new family of fluorescent probes with varying emission colors for selectively imaging hydrogen peroxide (H2O2) generated at physiological cell signaling levels. This structurally homologous series of fluorescein- and rhodol-based reporters relies on a chemospecific boronate-to-phenol switch to respond to H2O2 over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation and emission maxima and sensitivity to endogenously produced H2O2 signals, as shown by studies in RAW264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using one of the new H2O2-specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective discrimination between changes in H2O2 and hypochlorous acid (HOCl) levels in live RAW264.7 macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those that produced mainly H2O2, and those that possessed both types of ROS. The ability to monitor multiple ROS fluxes simultaneously using a palette of different colored fluorescent probes opens new opporunities to disentangle the complex contributions of oxidation biology to living systems by molecular imaging.