Recent advances in hydrogen peroxide imaging for biological applications

Abstract

Mounting evidence supports the role of hydrogen peroxide (H2O2) in physiological signaling as well as pathological conditions. However, the subtleties of peroxide-mediated signaling are not well understood, in part because the generation, degradation, and diffusion of H2O2 are highly volatile within different cellular compartments. Therefore, the direct measurement of H2O2 in living specimens is critically important. Fluorescent probes that can detect small changes in H2O2 levels within relevant cellular compartments are important tools to study the spatial dynamics of H2O2. To achieve temporal resolution, the probes must also be photostable enough to allow multiple readings over time without loss of signal. Traditional fluorescent redox sensitive probes that have been commonly used for the detection of H2O2 tend to react with a wide variety of reactive oxygen species (ROS) and often suffer from photostablilty issues. Recently, new classes of H2O2 probes have been designed to detect H2O2 with high selectivity. Advances in H2O2 measurement have enabled biomedical scientists to study H2O2 biology at a level of precision previously unachievable. In addition, new imaging techniques such as two-photon microscopy (TPM) have been employed for H2O2 detection, which permit real-time measurements of H2O2 in vivo. This review focuses on recent advances in H2O2 probe development and optical imaging technologies that have been developed for biomedical applications.

Keywords: Chemiluminescence; Fluorescence lifetime imaging microscopy (FLIM); Fluorescent probe; Hydrogen peroxide (H2O2); Molecular imaging; Nanoparticles; Ratiometric imaging; Reactive oxygen species (ROS); Two-photon microscopy.

Figure 1

Fluorescent turn-on mechanism and chemical structures of several examples of boronate-based H₂O₂ fluorescent probes. (A) Lactone-opening mechanism of fluorescence-enhancement for mono-boronate xanthene-based H₂O₂ probes. (B) Several examples of lactone-opening-based monoboronate H₂O₂ fluorescent probes.

Figure 2

Fluorescence imaging of intracellular H₂O₂ production using fluorescence probe PF6-AM (green). (A) Mechanism of Chemoselective H₂O₂ PF6-AM. (B) TPF imaging of H₂O₂ in astrocytes, fluorescence excited with a 770 nm Ti:sapphire laser. (C) Confocal microscopy of H₂O₂ in same astrocytes imaged in panel B, fluorescence excited with a 488 nm laser. The nuclei were stained with Hoechst 33342 (blue).

Figure 3

Ratiometric imaging of fresh rat hippocampal slice treated with H₂O₂. (A) The reaction between PN1 and H₂O₂ produced AN1 as the only major fluorescent product. (B) A hippocampal slice labeled with PN1. (C) Fluorescence spectra responses of 3 μM PN1 to 1 mM H₂O₂. Spectra were acquired at 0, 10, 20, 30, 40, 50, 60, and 120 min after H₂O₂ was added. (D) A hippocampal slice labeled with PN1 after pretreated with H2O2. Scale bars: 30 μm. The figures were adapted from ref. [50] with permission.

Figure 4

FLIM of HyPer-3 response to H₂O₂ production induced by inflammation in zebrafish larvae. (A) Left and right panels represent fluorescence intensity and FLIM images, respectively. ROI1 highlights the wound margin; ROI2 represents an area distant from the wound. (B) Fluorescence lifetime distribution plot for ROI1 and ROI2 in panel A. The figures were adapted from ref. [77] with permission.

Figure 5

In vivo imaging of H₂O₂ using peroxalate nanoparticles. (A) Peroxalate nanoparticle. (B) Chemiluminescence imaging. The figures were adapted from ref. [29] with permission.