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The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage.

Oeck S, Malewicz NM, Hurst S, Rudner J, Jendrossek V - Radiat Oncol (2015)

Bottom Line: It significantly reduced the analysis time of radiation-induced DNA-damage foci.The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis.As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the possibility to run all analyses in a batch mode.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Virchowstrasse 173, 45122, Essen, Germany. sebastian.oeck@uk-essen.de.

ABSTRACT

Background: The quantitative analysis of foci plays an important role in many cell biological methods such as counting of colonies or cells, organelles or vesicles, or the number of protein complexes. In radiation biology and molecular radiation oncology, DNA damage and DNA repair kinetics upon ionizing radiation (IR) are evaluated by counting protein clusters or accumulations of phosphorylated proteins recruited to DNA damage sites. Consistency in counting and interpretation of foci remains challenging. Many current software solutions describe instructions for time-consuming and error-prone manual analysis, provide incomplete algorithms for analysis or are expensive. Therefore, we aimed to develop a tool for costless, automated, quantitative and qualitative analysis of foci.

Methods: For this purpose we integrated a user-friendly interface into ImageJ and selected parameters to allow automated selection of regions of interest (ROIs) depending on their size and circularity. We added different export options and a batch analysis. The use of the Focinator was tested by analyzing γ-H2.AX foci in murine prostate adenocarcinoma cells (TRAMP-C1) at different time points after IR with 0.5 to 3 Gray (Gy). Additionally, measurements were performed by users with different backgrounds and experience.

Results: The Focinator turned out to be an easily adjustable tool for automation of foci counting. It significantly reduced the analysis time of radiation-induced DNA-damage foci. Furthermore, different user groups were able to achieve a similar counting velocity. Importantly, there was no difference in nuclei detection between the Focinator and ImageJ alone.

Conclusions: The Focinator is a costless, user-friendly tool for fast high-throughput evaluation of DNA repair foci. The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis. As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the possibility to run all analyses in a batch mode.

No MeSH data available.


Related in: MedlinePlus

The Focinator is a user friendly method that can be used without long term training. In Fig. 6, three different groups of users are compared. Programmers of the Focinator (n = 2), Biologist (n = 2) and users with no scientific background (n = 2) evaluated ten different cell lines. For each cell line about 80 pictures containing a total of about 500 nuclei were evaluated by the different users with the Focinator. The graph shows the calculated evaluation times per nucleus including a correction based on the numbers of pictures that had to be opened
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Fig6: The Focinator is a user friendly method that can be used without long term training. In Fig. 6, three different groups of users are compared. Programmers of the Focinator (n = 2), Biologist (n = 2) and users with no scientific background (n = 2) evaluated ten different cell lines. For each cell line about 80 pictures containing a total of about 500 nuclei were evaluated by the different users with the Focinator. The graph shows the calculated evaluation times per nucleus including a correction based on the numbers of pictures that had to be opened

Mentions: To prove the Focinator’s user-friendliness, the macro was tested by three different Focinator user groups, namely by the programmers of the Focinator (n = 2), by biologists (n = 2) and by users with no scientific background (n = 2) (Fig. 6). For the evaluation of applicability, all groups counted γ-H2.AX foci generated in response to different radiation doses in TRAMP-C1 cells at different time points post-irradiation using predefined parameters adjusted by an experienced scientist. In total 24,858 nuclei in 3361 images were counted. All users were able to use the Focinator after reading the software’s instruction manual. The data obtained by the programmers of the Focinator, the biologists and the users with no scientific background did not vary significantly in the mean evaluation times (Fig. 6). While the programmer needed 1.2 s per nucleus, the biologist needed 1.0, the users with no scientific background needed 1.54 s per nucleus (Fig. 6).Fig. 6


The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage.

Oeck S, Malewicz NM, Hurst S, Rudner J, Jendrossek V - Radiat Oncol (2015)

The Focinator is a user friendly method that can be used without long term training. In Fig. 6, three different groups of users are compared. Programmers of the Focinator (n = 2), Biologist (n = 2) and users with no scientific background (n = 2) evaluated ten different cell lines. For each cell line about 80 pictures containing a total of about 500 nuclei were evaluated by the different users with the Focinator. The graph shows the calculated evaluation times per nucleus including a correction based on the numbers of pictures that had to be opened
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4554354&req=5

Fig6: The Focinator is a user friendly method that can be used without long term training. In Fig. 6, three different groups of users are compared. Programmers of the Focinator (n = 2), Biologist (n = 2) and users with no scientific background (n = 2) evaluated ten different cell lines. For each cell line about 80 pictures containing a total of about 500 nuclei were evaluated by the different users with the Focinator. The graph shows the calculated evaluation times per nucleus including a correction based on the numbers of pictures that had to be opened
Mentions: To prove the Focinator’s user-friendliness, the macro was tested by three different Focinator user groups, namely by the programmers of the Focinator (n = 2), by biologists (n = 2) and by users with no scientific background (n = 2) (Fig. 6). For the evaluation of applicability, all groups counted γ-H2.AX foci generated in response to different radiation doses in TRAMP-C1 cells at different time points post-irradiation using predefined parameters adjusted by an experienced scientist. In total 24,858 nuclei in 3361 images were counted. All users were able to use the Focinator after reading the software’s instruction manual. The data obtained by the programmers of the Focinator, the biologists and the users with no scientific background did not vary significantly in the mean evaluation times (Fig. 6). While the programmer needed 1.2 s per nucleus, the biologist needed 1.0, the users with no scientific background needed 1.54 s per nucleus (Fig. 6).Fig. 6

Bottom Line: It significantly reduced the analysis time of radiation-induced DNA-damage foci.The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis.As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the possibility to run all analyses in a batch mode.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Virchowstrasse 173, 45122, Essen, Germany. sebastian.oeck@uk-essen.de.

ABSTRACT

Background: The quantitative analysis of foci plays an important role in many cell biological methods such as counting of colonies or cells, organelles or vesicles, or the number of protein complexes. In radiation biology and molecular radiation oncology, DNA damage and DNA repair kinetics upon ionizing radiation (IR) are evaluated by counting protein clusters or accumulations of phosphorylated proteins recruited to DNA damage sites. Consistency in counting and interpretation of foci remains challenging. Many current software solutions describe instructions for time-consuming and error-prone manual analysis, provide incomplete algorithms for analysis or are expensive. Therefore, we aimed to develop a tool for costless, automated, quantitative and qualitative analysis of foci.

Methods: For this purpose we integrated a user-friendly interface into ImageJ and selected parameters to allow automated selection of regions of interest (ROIs) depending on their size and circularity. We added different export options and a batch analysis. The use of the Focinator was tested by analyzing γ-H2.AX foci in murine prostate adenocarcinoma cells (TRAMP-C1) at different time points after IR with 0.5 to 3 Gray (Gy). Additionally, measurements were performed by users with different backgrounds and experience.

Results: The Focinator turned out to be an easily adjustable tool for automation of foci counting. It significantly reduced the analysis time of radiation-induced DNA-damage foci. Furthermore, different user groups were able to achieve a similar counting velocity. Importantly, there was no difference in nuclei detection between the Focinator and ImageJ alone.

Conclusions: The Focinator is a costless, user-friendly tool for fast high-throughput evaluation of DNA repair foci. The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis. As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the possibility to run all analyses in a batch mode.

No MeSH data available.


Related in: MedlinePlus