Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

Abstract

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.

In situ hybridization to detect DNA amplification in extracellular vesicles

“…An application note (https://oni.bio/oni-articles) suggested the possibility of visualizing generic EV DNA cargo (not a specific gene) by direct stochastic optical reconstruction microscopy (dSTORM, ONI). This elegant technique, available only a few centres, requires tedious set up and troubleshooting that hinders ease of applicability, which may explain the small number of publications to date that use this technology (Brunner et al, 2021;Mcnamara et al, 2022). Recently Yu et al summarized conventional and novel technologies for EVs isolation, characterization, and content detection, particularly in the context of EVs in liquid biopsy.…”

Quantitative dSTORM superresolution microscopy

“…However, the limited spatial resolution of confocal imaging precludes quantitative evaluation, because at high exposure dose the focus density is too high and therefore cluster analysis is not feasible. Localization data can be used to separate the nanofoci and open the way to quantitative evaluation [30]. However, the number of captured dSTORM images is limited.…”

Application of Lacunarity for Quantification of Single Molecule Localization Microscopy Images

“…In these studies the DSBs were artificially induced and visualized using the phosphorylated H2AX at Ser139 (referred to as γH2AX) as a double strand break marker in the nuclei of the cells [39]. The radiation treated U251, the neocarzinostatin (NCS) treated U2OS, and 4-hydroxytamoxifen (4-OHT) treated DIvA cell lines (U2OS cell line-based systems, which express and activate AsiSI homing endonuclease upon 4-OHT addition [40]) were studied in Brunner 2021 [32] and in Varga 2019 [21], respectively.…”

“…We believe that lacunarity analysis can be a competitive method for describing the structure of nanoscale cellular structures unraveled by single molecule localization microscopy. To demonstrate the effectiveness of lacunarity analysis, we revisited two of our previous quantitative dSTORM results [21,32] where cluster analysis played a central role in the evaluation process.…”