Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes

Abstract

Abstract. We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H 2 O 2 ) production in brain cells. For selective imaging of H 2 O 2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H 2 O 2 and mitochondria peroxy yellow 1 detects mitochondrial H 2 O 2 . Two-photon absorption cross sections for these H 2 O 2 probes are measured with a mode-locked Ti:sapphire laser in the wavelength range of 720 to 1040 nm. TPF imaging is demonstrated in the HT22 cell line to monitor both cytoplasmic H 2 O 2 and localized H 2 O 2 production in mitochondria. Endogenous cytoplasmic H 2 O 2 production is detected with TPF imaging in rat astrocytes modified with D-amino acid oxidase. The TPF H 2 O 2 imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H 2 O 2 . © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Spatial, Temporal, and Quantitative Manipulation of Intracellular Hydrogen Peroxide in Cultured Cells

“…We have used this model to manipulate H 2 O 2 production in astrocytes (Guo et al, 2013; Haskew-Layton et al, 2010). When R. gracilis DAAO expressing astrocytes are cocultured with neurons, we found that low concentrations of peroxide production (3.7 nmol/min/mg protein) protected neighboring neurons from glutamate-induced oxidative stress, thus showing that this model can be used to distinguish protective and nonprotective concentrations of peroxide (Haskew-Layton et al, 2010).…”

“…The use of conventional confocal microscopy to visualize fluorescence has limitations for real-time in vivo H 2 O 2 imaging, specifically phototoxicity, photo-bleaching, and limited imaging depth (Chen et al, 2013; Guo et al, 2013). In addition, prolonged light exposure can cause artifact ROS generation and signal amplification (Hockberger et al, 1999; Squirrell, Wokosin, White, & Bavister, 1999).…”

“…This method provides high-resolution (submicron) imaging with lower phototoxicity and deeper tissue penetration than confocal imaging (Chen et al, 2013; Guo et al, 2013). In the two-photon excitation model, a molecule simultaneously absorbs two photons whose individual energy is only half of the energy needed to excite that molecule, and then releases the energy to a fluorescence photon.…”

“…TPM imaging can be achieved from two-photon excitation of conventional fluorophores such as fluorescent dyes, fluorescent proteins, and nanoparticles. With fluorescent labeling, TPM has been used for intracellular imaging of molecules such as calcium, oxygen partial pressure ( p O 2 ), and ROS (Chen et al, 2013; Guo et al, 2013; Kwan & Dan, 2012; Sakadzic et al, 2010). It can also image endogenous fluorescence molecules such as the reduced nicotinamide adenine dinucleotide (NADH), FAD, and keratin (Chen et al, 2013; Zipfel et al, 2003).…”

“…This model used viral vectors to selectively express R. gracilis DAAO in astrocytes, followed by the subsequent application of exogenously applied D-alanine (D-ala) and flavin adenine dinucleotide (FAD) to produce H 2 O 2 (Haskew-Layton et al, 2010). The latter half of the chapter will discuss our validation method using two-photon microscopy (TPM) to detect site-specific fluorescent boronate probes sensitive to H 2 O 2 (Guo et al, 2013). The advantage of this method over other ROS detection methods is that it detects only intracellular H 2 O 2 concentrations (as opposed to other ROS).…”