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"Live Cell Imaging" at PTB’s micro ion beam

04.01.2010

The health risk caused by low radiation doses of less than 100 mSv can hardly be assessed because, on the one hand, epidemiological data exist for higher doses only and because, on the other hand, the radiation response can depend on many radiobiological effects as well as on individual sensitivities. Therefore, the radiation risk at low doses is currently the issue of the new research platform "Multidisciplinary European Low Dose Initiative" (MELODI) 1 of the EU and the national radiation protection organisations.

For a better assessment of the risk at low radiation doses, the basic biophysical and oncological mechanisms of action must be decoded. For the necessary experimental investigations, the microbeam of PTB is an ideal instrument. By means of a targeted irradiation with single or counted particles, well-defined radiation damage is generated in the cell nucleus or cytoplasm of living cells. In addition, the use of different ions and ion energies enables us to study the effects of different radiation qualities. For example, alpha particles with a high linear energy transfer (LET) of about 100 keV/µm are used to examine the impact of densely ionising particles as, e.g., in the case of radon decay. With energetic protons, however, the impact of loosely ionising X-radiation with a low LET of about 3 keV/µm to 20 keV/µm can be examined. In previous studies, mainly the late biological effects, such as clonal survival or chromosome aberrations due to false DNA repair, have been measured at the microbeam - and also in the neutron fields - of PTB.

In an interdisciplinary cooperation between PTB, the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH - DSMZ, Braunschweig) and the University Hospital of Düsseldorf, the new method of "live cell imaging" was established at the microbeam. Along particle tracks, double strand breaks (DSB) occur which - within seconds and minutes - trigger a variety of reactions and DNA repair processes in the cell. These initial responses can now be observed "live" as the appearance of fluorescent foci (along the particle tracks), because the cells have been genetically modified by fusing, for example, the green fluorescent protein (GFP) to a selected reporter or repair protein, which then accumulates at the DNA damage sites (see figure).

Within the cooperation, experiments with a selection of fluorescence-labelled repair proteins are carried out. The partners from the DSMZ and the University Hospital of Düsseldorf managed to engineer these fused proteins, insert them into a human cell line and to achieve stable expression. Thus, different processes and repair pathways can be selectively examined as a function of dose (number of particles), radiation quality and LET. In the areas of radiation protection and radiation therapy there are still open questions concerning the relative biological effectiveness (RBE) of particle radiation. Especially for the improvement of dosimetry concepts and for nano dosimetry, "live cell imaging" can make important contributions due to the quantification of the initial formation of DNA damage and the subsequent repair or false repair. At the same time, the cells’ reactions to UVA radiation are investigated with these labelled proteins at the DSMZ and at the University of Düsseldorf, to be able to detect, for example, similarities and differences in the DNA damage response.


prior to the irradiation


after the irradiation

Figure: Human fibroblasts (HT-1080) were stably transfected with the fluorescence-labelled reporter protein p53BP1. After the irradiation in a line pattern of 10 µm and an average distance of 1 µm between individual hits with alpha particles, the protein accumulates at double strand breaks which then become visible as luminous foci.

Bibliography

  1. www.hleg.de/fr.pdf