We present the synthesis, properties, and biological applications of Peroxy Lucifer 1 (PL1), a new fluorescent probe for imaging hydrogen peroxide produced in living cells by a ratiometric response. PL1 utilizes a chemoselective boronate-based switch to detect hydrogen peroxide by modulation of internal charge transfer (ICT) within a 1,8-naphthalimide dye. PL1 features high selectivity for hydrogen peroxide over similar reactive oxygen species, including superoxide, and nitric oxide, and a 65 nm shift in emission from blue-colored fluorescence to green-colored fluorescence upon reaction with peroxide. Two-photon confocal microscopy experiments in live macrophages show that PL1 can ratiometrically visualize localized hydrogen peroxide bursts generated in living cells at immune response levels.

Hydrogen peroxide (H2O2) is a potent small-molecule oxidant that can exert a diverse array of physiological and/or pathological effects within living systems depending on the timing and location of its production, accumulation, trafficking, and consumption. To help study the chemistry and biology of this reactive oxygen species (ROS) in its native cellular context, we now present a new method for monitoring local, subcellular changes in H2O2 levels by fluorescence imaging. Specifically, we have exploited the versatility of the SNAP-tag technology for site-specific protein labeling with small molecules on the surface or interior of living cells with the use of boronate-capped dyes to selectively visualize H2O2. The resulting SNAP-Peroxy-Green (SNAP-PG) probes consist of appropriately derivatized boronates bioconjugated to SNAP-tag fusion proteins. Spectroscopic measurements on the SNAP-PG constructs confirm their ability to detect H2O2 with specificity over other biologically relevant ROS. Moreover, these hybrid small-molecule/protein reporters can be used in live mammalian cells expressing SNAP-tag fusion proteins directed to the plasma membrane, nucleus, mitochondria, and endoplasmic reticulum. Imaging experiments using scanning confocal microscopy establish organelle-specific localization of the SNAP-tag probes and their fluorescence turn-on in response to changes in local H2O2 levels. This work provides a general molecular imaging platform for assaying H2O2 chemistry in living cells with subcellular resolution.

As the primary consumers of oxygen within all aerobic organisms, mitochondria are a major source of cellular reactive oxygen species (ROS) derived from the in vivo chemistry of oxygen metabolism. Mitochondrial ROS have been traditionally implicated in aging and in a variety of pathologies, including cancer, neurodegeneration, and diabetes, but recent studies also link controlled mitochondrial ROS fluxes to cell regulation and signaling events. Progress in the development of mitochondrial-targeted fluorescent small-molecule indicators that detect specific ROS with high selectivity offers a promising approach for interrogating mitochondrial ROS production, trafficking, and downstream biological effects.

We report a two-photon fluorescent probe (PN1) that can be excited by 750 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize H2O2 distribution in live cells and tissue by two-photon microscopy.