Automated detection of nuclei and DNA damage in Caenorhabditis elegans germline

The NUKE-BREAK macro for the program ImageJ (Schneider et al. 2012) was developed specifically to detect RAD-51 foci within nuclei in the C.elegans gonad, but can be applied to the detect and count any type of round particles. The macro was designed to automatically detect and process batches of microscope stacks (.tif) acquisitions of this organ. To this end, key parameters for nuclei and foci detection (minimum and maximum surface and circularity) have been optimized for the mitotic and meiotic regions of the gonad.

The macro creates a listing of .tif stacks to analyze from a root folder and processes them as follows:

1. The DAPI and RAD-51 staining channels are automatically assigned using preexisting BLUE and RED LUT.
2. The noise of both channel is then independently removed using the “substract background” function.
3. The DAPI channel is then used to detect the Gonad within the image by combining Z projections and the "Huang" thresholding method. A further analysis of the Gonad allows the automatic detection of its orientation and the position of its distal end. The position of every nuclei will be calculated using this reference point.
4. Nuclei are then detected by thresholding, for every slice, the pixel values present within the Gonad using the “Default” algorithm. As a nucleus is present on several slices, all particles having the same centroid are removed except of the one with the largest area value.
5. RAD-51 foci are then scored independently for each nucleus. In order to take into account the 3D shape of the nuclei, a Z projection is performed. RAD-51 foci are then detected within the shape of the nucleus using the “Max-Entropy” threshold method.
6. Finally, the program collects the position and size of all nuclei and foci to generate cumulative distribution curves over distance from the extremity of the gonad.

For more details please refer to our publication.

Detection of the gonad and nuclei	Raw nucleus vs RAD-51 foci and chromatine detection	Cumulative distribution of the foci along the gonad

Contributors

CLUET David	SIMBIO Team	[email protected]
MICHAL Fanny	EPIGENETIC REGULATION DURING DEVELOPMENT Team

Publication

The C. elegans SET-2/SET1 histone H3 Lys4 (H3K4) methyltransferase preserves genome stability in the germline.

M. Herbette, M.G. Mercier, F. Michal, D. Cluet, C. Burny, G. Yvert, V.J. Robert, F. Palladino

DNA Repair (Amst). 2017 Sep;57:139-150. doi: 10.1016/j.dnarep.2017.07.007. Epub 2017 Jul 29.

Author information:

Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon, Centre National de la Recherche Scientifique, Lyon, France.

License

Copyright CNRS 2013

This software is a computer program whose purpose is to automatically detect nuclei and DNA damage in Caenorhabditis elegans germline.

This software is governed by the CeCILL license under French law and abiding by the rules of distribution of free software. You can use, modify and/ or redistribute the software under the terms of the CeCILL license as circulated by CEA, CNRS and INRIA at the following URL: http://www.cecill.info/index.en.html

As a counterpart to the access to the source code and rights to copy, modify and redistribute granted by the license, users are provided only with a limited warranty and the software's author,the holder of the economic rights, and the successive licensors have only limited liability.

In this respect, the user's attention is drawn to the risks associated with loading, using, modifying and/or developing or reproducing the software by the user in light of its specific status of free software, that may mean that it is complicated to manipulate, and that also therefore means that it is reserved for developers and experienced professionals having in-depth computer knowledge. Users are therefore encouraged to load and test the software's suitability as regards their requirements in conditions enabling the security of their systems and/or data to be ensured and, more generally, to use and operate it in the same conditions as regards security.

The fact that you are presently reading this means that you have had knowledge of the CeCILL license and that you accept its terms.

Requirements

The NUKE-BREAK macro requires ImageJ v1.49g or higher (Download).

Files

• [] src
  • README.md
  • LICENSE
  • Installation.ijm
  • Installation_FIJI.ijm
  • [] doc
    • FIJI.jpg
    • IJ.jpg
    • Logo_cnrs.jpg
    • Logo_ens.jpg
    • Logo_LBMC.jpg
  • [] macro
    • BreakFinder.java
    • Cleaner.java
    • CNRS.jpg
    • createSettings.java
    • ENS.jpg
    • Explorer.java
    • GonadeDetector.java
    • HTML_Nuke.html
    • LBMC.jpg
    • Main.java
    • NucleiDetector.java
    • openSettings.java
    • Reconstruction.java
    • ROIeraser.java
    • Settings.txt
    • Startup_CL.txt
    • Startup_CL2.txt
    • style_Nuke.css
    • Table_Analysis.java
    • Table.java
    • tableline.html
    • Twinkiller.java
    • UCBL.jpg

Installation

The NUKE-BREAK macro can be automatically installed with all required files in ImageJ and FIJI. Please follow the specific instructions described below.

1. Open ImageJ.
2. Open the src folder of the NUKE-BREAK macro.
3. Drag the Installation.ijm file on ImageJ Menu bar to open it.
4. In the Menu bar of the macro select the Macros/Run Macro option.
5. The window will be closed automatically and all required files will be installed in the ImageJ/macros/NUKE-BREAK folder. The shortcut Plugins/Macros/NUKE-BREAK will be added in the Menu bar.
6. A second shortcut ImageJ/macros/NUKE-BREAK_Table is generated to allow the generation of cumulative curve after a human quality control (if required).
7. Restart ImageJ to refresh the Menu bar.

1. Open FIJI.
2. Open the src folder of the NUKE-BREAK macro.
3. Drag the Installation_Fiji.ijm file on FIJI Menu bar to open it.
4. In the console select the Run option.
5. All required files will be installed in the Fiji.app/macros/NUKE-BREAK folder. The shortcut Plugins/Macros/NUKE-BREAK will be added in the Menu bar.
6. 6. A second shortcut ImageJ/macros/NUKE-BREAK_Table is generated to allow the generation of cumulative curve after a human quality control (if required).
7. Restart FIJI to refresh the Menu bar.

Update

Follow the same instructions as for the installation process.