title: Fluorescence imaging of reactive oxygen species by confocal laser scanning microscopy for track analysis of synchrotron X-ray photoelectric nanoradiator dose: X-ray pump-optical probe.
abstract: Bursts of emissions of low-energy electrons, including interatomic Coulomb decay electrons and Auger electrons (0-1000 eV), as well as X-ray fluorescence produced by irradiation of large-Z element nanoparticles by either X-ray photons or high-energy ion beams, is referred to as the nanoradiator effect. In therapeutic applications, this effect can damage pathological tissues that selectively take up the nanoparticles. Herein, a new nanoradiator dosimetry method is presented that uses probes for reactive oxygen species (ROS) incorporated into three-dimensional gels, on which macrophages containing iron oxide nanoparticles (IONs) are attached. This method, together with site-specific irradiation of the intracellular nanoparticles from a microbeam of polychromatic synchrotron X-rays (5-14 keV), measures the range and distribution of OH radicals produced by X-ray emission or superoxide anions ({\rm{O}}_2^-) produced by low-energy electrons. The measurements are based on confocal laser scanning of the fluorescence of the hydroxyl radical probe 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) or the superoxide probe hydroethidine-dihydroethidium (DHE) that was oxidized by each ROS, enabling tracking of the radiation dose emitted by the nanoradiator. In the range 70 µm below the irradiated cell, ^\bullet{\rm{OH}} radicals derived mostly from either incident X-ray or X-ray fluorescence of ION nanoradiators are distributed along the line of depth direction in ROS gel. In contrast, {\rm{O}}_2^- derived from secondary electron or low-energy electron emission by ION nanoradiators are scattered over the ROS gel. ROS fluorescence due to the ION nanoradiators was observed continuously to a depth of 1.5 mm for both oxidized APF and oxidized DHE with relatively large intensity compared with the fluorescence caused by the ROS produced solely by incident primary X-rays, which was limited to a depth of 600 µm, suggesting dose enhancement as well as more penetration by nanoradiators. In conclusion, the combined use of a synchrotron X-ray microbeam-irradiated three-dimensional ROS gel and confocal laser scanning fluorescence microscopy provides a simple dosimetry method for track analysis of X-ray photoelectric nanoradiator radiation, suggesting extensive cellular damage with dose-enhancement beyond a single cell containing IONs.
authors: Department of Biomedical Engineering, School of Medicine, Catholic University of Daegu, Daegu 42472, Republic of Korea.
authors: Jae Kun
authors: JK
authors: Jeon
authors: Department of Anatomy, School of Medicine, Catholic University of Daegu, Daegu 42472, Republic of Korea.
authors: Sung Mi
authors: SM
authors: Han
authors: Department of Biomedical Engineering, School of Medicine, Catholic University of Daegu, Daegu 42472, Republic of Korea.
authors: Jong Ki
authors: JK
authors: Kim
keywords: ROS fluorescence; X-ray photoelectric; confocal laser scanning; gel dosimetry; nanoradiator effect