Fig. 2.

Induction of DNA double-strand breaks by multi-microbeam irradiation. a MMB irradiation induces DSB responses. U2OS cells were irradiated for 6 min through a metal mesh filter with openings of 2.2 μm in diameter, fixed 30 min later, and stained for DNA (blue), γH2AX (red), and Rad51 (green, left panel) or MDC1 (green, right panel). Inset shows magnification of a single irradiated area. Scale bar 10 μm. b The number of DSBs induced by MMB irradiation depends linearly on irradiation time. Left panels show U2OS cells irradiated for 5, 10, or 15 s through a metal mesh filter with openings of 2.2 μm in diameter, fixed 5 min later, and stained for DNA (blue), γH2AX (red), and MDC1 (green). Right panel shows numbers of γH2AX foci representing individual DSBs per exposed area in cells from b. c The geometry of exposed areas can be controlled by applying metal mesh filters of various parameters. Cells were irradiated for 6 min using filters with openings of 2.2 μm (left) or 3.5 μm in diameter (right), fixed 5 min later, and stained for DNA (blue) and γH2AX (red). Upper panels show mesh filters used, imaged by a scanning electron microscope. Lower panels show stained cells irradiated using the respective filters. Scale bar 2.2 μm (left panel) and 3.5 μm (right panel)