Abstract
We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H&sub2;O&sub2;) production in brain cells. For selective imaging of H&sub2;O&sub2; 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&sub2;O&sub2; and mitochondria peroxy yellow 1 detects mitochondrial H&sub2;O&sub2;. Two-photon absorption cross sections for these H&sub2;O&sub2; 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&sub2;O&sub2; and localized H&sub2;O&sub2; production in mitochondria. Endogenous cytoplasmic H&sub2;O&sub2; production is detected with TPF imaging in rat astrocytes modified with d-amino acid oxidase. The TPF H&sub2;O&sub2; imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H&sub2;O&sub2;.