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Updated: May 7, 2026
Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining
Published on: June 23, 2020
Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes
Hengchang Guo1, Hossein Aleyasin, Scott S Howard
1University of Maryland, Fischell Department of Bioengineering, College Park, Maryland 20742bWeill Medical College of Cornell University, Burke Medical Research Institute, White Plains, New York 10605.
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.
