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

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. (C) The Authors.