High-resolution µCT of a mouse embryo using a compact laser-driven X-ray betatron source.

In the field of X-ray microcomputed tomography (µCT) there is a growing need to reduce acquisition times at high spatial resolution (approximate micrometers) to facilitate in vivo and high-throughput operations. The state of the art represented by synchrotron light sources is not practical for certain applications, and therefore the development of high-brightness laboratory-scale sources is crucial. We present here imaging of a fixed embryonic mouse sample using a compact laser-plasma-based X-ray light source and compare the results to images obtained using a commercial X-ray µCT scanner. The radiation is generated by the betatron motion of electrons inside a dilute and transient plasma, which circumvents the flux limitations imposed by the solid or liquid anodes used in conventional electron-impact X-ray tubes. This X-ray source is pulsed (duration <30 fs), bright (>1010 photons per pulse), small (diameter <1 µm), and has a critical energy >15 keV. Stable X-ray performance enabled tomographic imaging of equivalent quality to that of the µCT scanner, an important confirmation of the suitability of the laser-driven source for applications. The X-ray flux achievable with this approach scales with the laser repetition rate without compromising the source size, which will allow the recording of high-resolution µCT scans in minutes.

Super-Resolution Nanoscopy Imaging Applied to DNA Double-Strand Breaks.

Genomic deoxyribonucleic acid (DNA) is continuously being damaged by endogenous processes such as metabolism or by exogenous events such as radiation. The specific phosphorylation of histone H2AX on serine residue 139, described as γ-H2AX, is an excellent indicator or marker of DNA double-strand breaks (DSBs). The yield of γ-H2AX (foci) is shown to have some correlation with the dose of radiation or other DSB-causing agents. However, there is some discrepancy in the DNA DSB foci yield among imaging and other methods such as gel electrophoresis. Super-resolution imaging techniques are now becoming widely used as essential tools in biology and medicine, after a slow uptake of their development almost two decades ago. Here we compare several super-resolution techniques used to image and determine the amount and spatial distribution of γ-H2AX foci formation after X-ray irradiation: stimulated emission depletion (STED), ground-state depletion microscopy followed by individual molecule return (GSDIM), structured illumination microscopy (SIM), as well as an improved confocal, Airyscan and HyVolution 2. We show that by using these super-resolution imaging techniques with as low as 30-nm resolution, each focus may be further resolved, thus increasing the number of foci per radiation dose compared to standard microscopy. Furthermore, the DNA repair proteins 53BP1 (after low-LET irradiations) and Ku70/Ku80 (from laser microbeam irradiation) do not always yield a significantly increased number of foci when imaged by the super-resolution techniques, suggesting that γ-H2AX, 53PB1 and Ku70/80 repair proteins do not fully co-localize on the units of higher order chromatin structure.