Figure 5

Targeted irradiation of fluorescence labeled chromatin domains. A TexasRed labeled nucleotide analogue was introduced via electroporation into Hela cells already stably transfected with a plasmid coding for a MDC1-GFP fusion protein (green). When present during S-phase of the cell cycle, the labeled nucleotide is incorporated into the DNA. After 72 h (2–3 cell divisions) separate chromatin domains (red) can be detected. One of these was chosen to be irradiated with a single carbon ion as the center of a nine point cross like pattern. The figure shows MDC1-GFP accumulation one minute after irradiation at the sites of DNA damage, which reflects nicely the irradiation pattern and gives the possibility to check if targeting was successful. In the left panel of the figure the irradiated cells are shown, in the right panel additionally the intended target is shown as white dot and the actual center of the irradiated cross-wise pattern is indicated by the blue dot. Note that we explain the strong red staining at the rim of cell 6 by conglomeration of TexasRed or TexasRed-dUTP. The ring-like structure suggests that the signal is located out-of-focus of the microscopic image, i.e. above or below the cell.