Selective high-dose irradiation and PCNA recruitment. (A) Target definition prior to irradiation in three cells selected from a whole microscopic field (see Additional file 1). Cells are to be irradiated with a five point cross with approximately 1000 carbon ions per point (each point was irradiated for 1 second with a particle fluence of about 1 kHz). For better illustration of the foci recruitment kinetics, indicated times refer to the start of the irradiation of the upper left cell, not to the irradiation start of the whole field of view. After the irradiation of the upper left cell, a cell not shown here was targeted (compare Additional file 1), followed by the lower left and the lower right cell. Thus irradiation of these cells took in total 20 seconds. Image acquisition started before irradiation. One second after irradiation start of the first cell (during irradiation of the second point in the first cell), no PCNA accumulation can be detected (B). Three seconds later, while irradiation of the first cell is still in progress, already two sites of PCNA accumulation are clearly detectable (C), Ongoing PCNA recruitment within the three nuclei (D-H). See Figure 4 for a detailed analysis of the foci recruitment. Comparing the intensities of the foci of the upper left cell shows ongoing PCNA accumulation for at least 35 seconds (C-H). PCNA protein is visualized using the “Cell Cycle Chromobody plasmid” (ChromoTek, Germany).