Abstract
We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H₂O₂) production in brain cells. For selective imaging of H₂O₂ 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 H₂O₂ and mitochondria peroxy yellow 1 detects mitochondrial H₂O₂. Two-photon absorption cross sections for these H₂O₂ 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 H₂O₂ and localized H₂O₂ production in mitochondria. Endogenous cytoplasmic H₂O₂ production is detected with TPF imaging in rat astrocytes modified with d-amino acid oxidase. The TPF H₂O₂ imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H₂O₂.
Highlights
IntroductionHydrogen peroxide (H2O2), a common reactive oxygen species (ROS) found in biological systems, is recognized as an intracellular second messenger for cellular signaling that exerts diverse physiological and pathological effects.[1,2,3,4,5,6,7] The aberrant production or accumulation of H2O2 within cellular mitochondria over time due to oxidative stress or genetic mutation is connected to serious pathological conditions including cancer,[8] diabetes,[9] and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, as well as stroke.[10,11,12] In addition, H2O2 is involved in therapeutic processes such as wound healing, stem cell proliferation, and an adaptive response in astrocytes leading to neuronal protection.[1,2,7,13]A substantial challenge in elucidating the diverse roles of H2O2 in complex biological environments is the lack of methods to determine the spatial and temporal dynamics of this reactive oxygen metabolite in living systems
mitochondria peroxy yellow 1 (MitoPY1) [Fig. 1(d)] was derived from peroxy yellow 1 (PY1) to include a combination of a boronate-based switch and a mitochondrial-targeting phosphonium moiety for the detection of H2O2 localized to cellular mitochondria.[18]
The peak cross section values of chemoselective probes are comparable to that of fluorescein,[28] which is sufficiently large for two-photon imaging in vitro or in vivo
Summary
Hydrogen peroxide (H2O2), a common reactive oxygen species (ROS) found in biological systems, is recognized as an intracellular second messenger for cellular signaling that exerts diverse physiological and pathological effects.[1,2,3,4,5,6,7] The aberrant production or accumulation of H2O2 within cellular mitochondria over time due to oxidative stress or genetic mutation is connected to serious pathological conditions including cancer,[8] diabetes,[9] and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, as well as stroke.[10,11,12] In addition, H2O2 is involved in therapeutic processes such as wound healing, stem cell proliferation, and an adaptive response in astrocytes leading to neuronal protection.[1,2,7,13]A substantial challenge in elucidating the diverse roles of H2O2 in complex biological environments is the lack of methods to determine the spatial and temporal dynamics of this reactive oxygen metabolite in living systems. For the detection of ROS production in vitro, several fluorescent probes have been developed based on small molecules, fluorescent proteins, and conventional confocal microscopy has limitations for use in real time in vivo H2O2 imaging, including photodamage, photobleaching, and limited imaging depth. Prolonged visible light exposure can result in artifactual ROS generation and signal amplification.[23,24] two-photon imaging of H2O2 offers an attractive alternative to overcome many of these limitations.[25,26,27] We report two-photon fluorescence (TPF) imaging for the detection of intracellular cytoplasmic
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