The clonogenic cell survival assay is a basic method to study the cytotoxic effect of radiation and chemical toxins. In large experimental setups, counting of colonies by eye is tiresome and prone to bias. Moreover, it is often interesting to quantify the size of individual colonies. Such analyses are largely facilitated by computerised image analysis systems. Although a number of such systems exist, they all focus on enumerating colonies and not on analysing the colony size. We have developed a new software package for both counting colonies and plotting their size distributions. The software called count and Plot HIstograms of Colony Size (countPHICS) consists of two parts: (1) a macro written for ImageJ which analyses computerised images of cell culture dishes or 6-well plates, counts colonies, estimates their size and saves the results in a text file; (2) a program written with QT Creator which reads the text file, plots histograms of colony size distribution and fits the best function. The full program is freely available at:
http://www.fuw.edu.pl/~bbrzozow/FizMed/countPHICS.html
. In conclusion, our new publically available software will facilitate colony counting and provide additional information on the colony growth rate, which is relevant especially for radiosensitisation studies.
Ionizing radiation induces a variety of DNA lesions, including single and double strand breaks. Large energy deposition precisely localized along the ion track that occurs in the case of heavy ion irradiation can lead to complex types of DNA double strand breaks in exposed biological material. The formation of nuclear double strand breaks triggers phosphorylation of histone H2AX, which can be microscopically visualized as foci in the γ-H2AX assay. Studies with a carbon ion beam are being carried out at the Heavy Ion Laboratory of the University of Warsaw. The γ-H2AX assay as a method of measuring the biological response of cells irradiated with 12 C ions as well as the frequency cluster size distributions obtained in the nanodosimetry experiment at HIL will be presented.
The irradiation system consisting of an α-source and disc holder has been developed in the Heavy Ion Laboratory, University of Warsaw. A simple exposure system for irradiation of biological samples consists of the Am-241 disc source, source holder and biological samples cultured in special Petri dishes. The irradiation system has been investigated to determine the alpha spectrum and dose distribution in irradiated single cell layer attached to the Mylar foil. Commercial Am-241 disc source of 50 mm in diameter, with a radioactive element embedded into a substrate layer was examined to established the uniformity of surface radioactivity over the disc source. The experimental device is equipped with cell dishes of 40 mm in diameter and a 6 µm thick Mylar foil bottom. Care was taken for homogenous irradiation of the cells. Dose calibration for the irradiation system was calculated taking into account source-to-target geometry.
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