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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’s accuracy is comparable to manual counting and evaluation only with ImageJ. ImageJ-based, manual counting and the usage of the Focinator macro were compared. To evaluate the repair time-dependent decrease of γ-H2.AX foci after irradiation TRAMP-C1 cells were irradiated with 3 Gy, incubated at 37 °C and fixed 0.5, 1, 2, 4, 6 and 24 h after irradiation. The cells were permeabilized and stained with an Alexa 647-linked anti- γ-H2.AX antibody. A total number of approximately 40 nuclei per time point was evaluated. a Development of the mean foci count per nucleus form three independent experiments at stated time points after irradiation. b A dose response curve depicts foci count after different doses (0.5, 1.5 and 3 Gy) 30 min after irradiation. A direct correlation between the different scoring methods with respective correlations value (R2) at the time points 0.5, 1, 2, 4, 6 and 24 h after irradiation is shown for Focinator-based evaluation in comparison to using ImageJ alone in (c) and compared to manual counting in (d)
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Fig5: The Focinator’s accuracy is comparable to manual counting and evaluation only with ImageJ. ImageJ-based, manual counting and the usage of the Focinator macro were compared. To evaluate the repair time-dependent decrease of γ-H2.AX foci after irradiation TRAMP-C1 cells were irradiated with 3 Gy, incubated at 37 °C and fixed 0.5, 1, 2, 4, 6 and 24 h after irradiation. The cells were permeabilized and stained with an Alexa 647-linked anti- γ-H2.AX antibody. A total number of approximately 40 nuclei per time point was evaluated. a Development of the mean foci count per nucleus form three independent experiments at stated time points after irradiation. b A dose response curve depicts foci count after different doses (0.5, 1.5 and 3 Gy) 30 min after irradiation. A direct correlation between the different scoring methods with respective correlations value (R2) at the time points 0.5, 1, 2, 4, 6 and 24 h after irradiation is shown for Focinator-based evaluation in comparison to using ImageJ alone in (c) and compared to manual counting in (d)

Mentions: For validation of the Focinator, TRAMP-C1 cells were irradiated with 3 Gy and foci were analyzed before and 0.5, 1, 2, 4, 6 and 24 h after irradiation. Again, the results of the Focinator were compared to ImageJ-based analysis and manual counting. All three methods showed a uniform time-dependent decrease of γ-H2.AX foci after the initial maximum at 30 min post-irradiation, proving the validity of the developed macro for reliable foci-counting (Fig. 5a). Counting of foci after irradiating cells with different doses (0.5, 1.5 and 3 Gy; 30 min after irradiation) was performed to validate the use of the Focinator at different amounts of DNA damage. The dose response curve shows a linear relationship between the number of foci per cell and the clinically relevant radiation doses used in the present study, thereby concurring with previously published literature [35–38]. Moreover, there was a strong similarity of foci numbers counted in 439 nuclei manually or with the Focinator (Fig. 5c, R2 = 0.9670). The comparison of ImageJ-based analysis with the Focinator achieved also high correlation indicating that the results of both analysis programs were very similar (Fig. 5d, R2 = 0.9914). Though a slight underestimation of counted foci was observed one hour after irradiation when using ImageJ and the Focinator compared to manual analysis, this effect was not significant. A similar phenomenon has previously been described by others and has been attributed to the increasing amount of overlapping foci at high foci numbers per nucleus, as well as at high irradiation doses and shorter repair times yielding increased foci size [12]. However, it has been suggested that the falsification of the results by high numbers of overlapping foci can be minimized when considering an additional analysis of foci intensity [25]. In contrast to manual analysis, the Focinator provides the opportunity to quantify the nuclei size as well as the minimal, mean and maximal intensity of the foci and the nuclei, and is thus superior to manual analysis. Another advantage of the Focinator is the opportunity to measure the ROIs area size. Accordingly, foci can be counted per area and not only per nucleus.Fig. 5


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’s accuracy is comparable to manual counting and evaluation only with ImageJ. ImageJ-based, manual counting and the usage of the Focinator macro were compared. To evaluate the repair time-dependent decrease of γ-H2.AX foci after irradiation TRAMP-C1 cells were irradiated with 3 Gy, incubated at 37 °C and fixed 0.5, 1, 2, 4, 6 and 24 h after irradiation. The cells were permeabilized and stained with an Alexa 647-linked anti- γ-H2.AX antibody. A total number of approximately 40 nuclei per time point was evaluated. a Development of the mean foci count per nucleus form three independent experiments at stated time points after irradiation. b A dose response curve depicts foci count after different doses (0.5, 1.5 and 3 Gy) 30 min after irradiation. A direct correlation between the different scoring methods with respective correlations value (R2) at the time points 0.5, 1, 2, 4, 6 and 24 h after irradiation is shown for Focinator-based evaluation in comparison to using ImageJ alone in (c) and compared to manual counting in (d)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: The Focinator’s accuracy is comparable to manual counting and evaluation only with ImageJ. ImageJ-based, manual counting and the usage of the Focinator macro were compared. To evaluate the repair time-dependent decrease of γ-H2.AX foci after irradiation TRAMP-C1 cells were irradiated with 3 Gy, incubated at 37 °C and fixed 0.5, 1, 2, 4, 6 and 24 h after irradiation. The cells were permeabilized and stained with an Alexa 647-linked anti- γ-H2.AX antibody. A total number of approximately 40 nuclei per time point was evaluated. a Development of the mean foci count per nucleus form three independent experiments at stated time points after irradiation. b A dose response curve depicts foci count after different doses (0.5, 1.5 and 3 Gy) 30 min after irradiation. A direct correlation between the different scoring methods with respective correlations value (R2) at the time points 0.5, 1, 2, 4, 6 and 24 h after irradiation is shown for Focinator-based evaluation in comparison to using ImageJ alone in (c) and compared to manual counting in (d)
Mentions: For validation of the Focinator, TRAMP-C1 cells were irradiated with 3 Gy and foci were analyzed before and 0.5, 1, 2, 4, 6 and 24 h after irradiation. Again, the results of the Focinator were compared to ImageJ-based analysis and manual counting. All three methods showed a uniform time-dependent decrease of γ-H2.AX foci after the initial maximum at 30 min post-irradiation, proving the validity of the developed macro for reliable foci-counting (Fig. 5a). Counting of foci after irradiating cells with different doses (0.5, 1.5 and 3 Gy; 30 min after irradiation) was performed to validate the use of the Focinator at different amounts of DNA damage. The dose response curve shows a linear relationship between the number of foci per cell and the clinically relevant radiation doses used in the present study, thereby concurring with previously published literature [35–38]. Moreover, there was a strong similarity of foci numbers counted in 439 nuclei manually or with the Focinator (Fig. 5c, R2 = 0.9670). The comparison of ImageJ-based analysis with the Focinator achieved also high correlation indicating that the results of both analysis programs were very similar (Fig. 5d, R2 = 0.9914). Though a slight underestimation of counted foci was observed one hour after irradiation when using ImageJ and the Focinator compared to manual analysis, this effect was not significant. A similar phenomenon has previously been described by others and has been attributed to the increasing amount of overlapping foci at high foci numbers per nucleus, as well as at high irradiation doses and shorter repair times yielding increased foci size [12]. However, it has been suggested that the falsification of the results by high numbers of overlapping foci can be minimized when considering an additional analysis of foci intensity [25]. In contrast to manual analysis, the Focinator provides the opportunity to quantify the nuclei size as well as the minimal, mean and maximal intensity of the foci and the nuclei, and is thus superior to manual analysis. Another advantage of the Focinator is the opportunity to measure the ROIs area size. Accordingly, foci can be counted per area and not only per nucleus.Fig. 5

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