Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

The quantitative detection of radiation caused DNA double-strand breaks (DSB) by immunostained γ-H2AX foci using direct stochastic optical reconstruction microscopy (dSTORM) provides a deeper insight into the DNA repair process at nanoscale in a time-dependent manner. Glioblastoma (U251) cells were irradiated with 250 keV X-ray at 0, 2, 5, 8 Gy dose levels. Cell cycle phase distribution and apoptosis of U251 cells upon irradiation was assayed by flow cytometry. We studied the density, topology and volume of the γ-H2AX foci with 3D confocal microscopy and the dSTORM superresolution method. A pronounced increase in γ-H2AX foci and cluster density was detected by 3D confocal microscopy after 2 Gy, at 30 min postirradiation, but both returned to the control level at 24 h. Meanwhile, at 24 h a considerable amount of residual foci could be measured from 5 Gy, which returned to the normal level 48 h later. The dSTORM based γ-H2AX analysis revealed that the micron-sized γ-H2AX foci are composed of distinct smaller units with a few tens of nanometers. The density of these clusters, the epitope number and the dynamics of γ-H2AX foci loss could be analyzed. Our findings suggest a discrete level of repair enzyme capacity and the restart of the repair process for the residual DSBs, even beyond 24 h. The dSTORM superresolution technique provides a higher precision over 3D confocal microscopy to study radiation induced γ-H2AX foci and molecular rearrangements during the repair process, opening a novel perspective for radiation research

Spectral and spatial shaping of laser-driven proton beams using a pulsed high-field magnet beamline

Intense laser-driven proton pulses, inherently broadband and highly divergent, pose a challenge to established beamline concepts on the path to application-adapted irradiation field formation, particularly for 3D. Here we experimentally show the successful implementation of a highly efficient (50% transmission) and tuneable dual pulsed solenoid setup to generate a homogeneous (8.5% uniformity laterally and in depth) volumetric dose distribution (cylindrical volume of 5 mm diameter and depth) at a single pulse dose of 0.7 Gy via multi-energy slice selection from the broad input spectrum. The experiments have been conducted at the Petawatt beam of the Dresden Laser Acceleration Source Draco and were aided by a predictive simulation model verified by proton transport studies. With the characterised beamline we investigated manipulation and matching of lateral and depth dose profiles to various desired applications and targets. Using a specifically adapted dose profile, we successfully performed first proof-of-concept laser-driven proton irradiation studies of volumetric in-vivo normal tissue (zebrafish embryos) and in-vitro tumour tissue (SAS spheroids) samples.Comment: Submitted to Scientific Report

Novel realtime cell analysis platform for the dynamic monitoring of ionizing radiation effects on human tumor cell lines and primary fibroblasts

Translational research in radiation oncology is important for the detection of adverse radiation effects, cellular responses, and radiation modifications, and may help to improve the outcome of radiation therapy in patients with cancer. The present study aimed to optimize and validate a realtime labelfree assay for the dynamic monitoring of cellular responses to ionizing radiation. The xCELLigence system is an impedancebased platform that provides continuous information on alterations in cell size, shape, adhesion, proliferation, and survival. In the present study, various malignant human primary fibroblast cells (U251, GBM2, MCF7, A549, HT29) were exposed to 0, 5 and 10 Gy of Cobalt60 radiation. As well as the xCELLigence system, cell survival and proliferation was evaluated using the following conventional endpoint cellbased methods: Clonogenic, MTS, and lactate dehydrogenase assays, and apoptosis was detected by fluorescenceactivated cell sorting. The effects of ionizing radiation were detected for each cell line using impedance monitoring. The realtime data correlated with the colony forming assay results. At low cell densities (1,0002,000 cells/well) the impedancebased method was more accurate at monitoring dosedependent changes in the malignant human primary fibroblast cell lines, as compared with the endpoint assays. The results of the present study demonstrated that the xCELLigence system may be a reliable and rapid diagnostic method for the monitoring of dynamic cell behavior following radiation. In addition, the xCELLigence system may be used to investigate the cellular mechanisms underlying the radiation response, as well as the timedependent effects of radiation on cell proliferation and viability