Automated Detection of Proximity Ligation Assay

Introduction

The PLA macro for the program ImageJ (Schneider et al. 2012) is based on a previously released macro (NUKE-BREAK, Herbette et al. 2017) and optimized to detect Proximity Ligation Assay foci within nuclei of human cells. The macro was designed to automatically detect and process batches of microscope coupled acquisitions. To this end, key parameters for nuclei and foci detection (minimum and maximum surface and circularity) have been optimized.

The PLA macro explores recursively a specific folder and treat all images with the correct extension (chosen by the user).

ORIGINAL	TREATED

The macro will generate a listing of couples of acquisition images using user defined DAPI.extension and PLA.extension. Using the GUI, the user can specify the extensions as the main analysis parameters:

• Minimum Surface for initial nuclei detection
• Maximum Surface for initial nuclei detection
• Maximum Surface of a single nucleus (for nuclei aggregates identification and splitting)
• Minimum Circularity of single nucleus (for nuclei aggregates identification and splitting)
• Minimum Surface for PLA foci
• Maximum Surface for PLA foci

These parameters can be changed and saved, so the program can be adapted to specific acquisition resolution and/or experimental conditions affecting the geometry of the nuclei and the PLA foci.

The images couples are then processed as follows:

1. The noise of both DAPI and PLA images is independently removed using the “substract background” function.
2. The DAPI channel is then used to detect the Nuclei with the "Huang" thresholding method and the initial Nuclei Minimum surface and Maximum surface parameters.
3. The aggregates of nuclei are subsequently identified using the single Nucleus Maximum surface and Minimum Circularity. Detected ROIs fitting with these parameters are then splitted using the "Watershed" algorithm.
4. PLA foci are then detected independently for every isolated nucleus, using the “Max-Entropy” threshold method and the foci Minimum Surface and Maximum Surface parameters.
5. Finally, the program collects the size of all nuclei and foci to generate a Results.csv table (see below).

Input

For each aquistion 2 files should be provided:

• One DAPI (or any other nucleus labelling) staining.
• One PLA staining

All DAPI files should have the same explicit extension (e.g. _w1DAPI.TIF). It should be the same for the PLA files (e.g. _w2RFP.TIF). Only DAPI files with a corresponding PLA file will be treated.

As the program is able to treat files as batch, all images must have the same resolution.

Output

The macro generate several files and folders that have all the same Fingerprint (Date and time).

• 1 yyyy-mm-dd_hh-mm_Parameters_and_Files.txt file in the root folder indicated by the user. This file contains all parameters used for the analysis, and the list of all the picture files that the macro will treat.
• 1 yyyy-mm-dd_hh-mm_Results.csv table file containing the properties of all Nuclei and PLA Foci detected for all images. The data are presented in the following columns:
  • File: File name.
  • Nuclei: Names of the Nuclei in the file.
  • Surface: Surface in pixels of the Nuclei.
  • PLA foci: Names of PLA Foci in one Nucleus.
  • Surface PLA: Total PLA surface in a Nucleus or Surface of each PLA Foci.
  • % of Nucleus surface: Percentage of the surface of a Nucleus covered by the total PLA Foci, or by each PLA Foci.
• 1 yyyy-mm-dd_hh-mm_ImageName folder containing:
  • 1 ImageName_Bilan.jpg file showing the identified Nuclei and PLA Foci on the original DAPI and PLA stainings.
  • 1 ImageName_Nuclei.jpg file showing the identified Nuclei on the original DAPI staining.
  • 1 ImageName_PLA.jpg file showing the identified Nuclei and associated PLA Foci on the original PLA staining.
  • 1 ImageName_Original.jpg file presenting the overlay of original DAPI and PLA stainings input.
  • 1 ImageName_Nuclei.zip ROIset file for ImageJ. This is the annotated ROIs of the detected Nuclei.
  • 1 ImageName_Nuclei-PLA.zip ROIset file for ImageJ. This is the annotated ROIs of the detected Nuclei directly associated with the ROIs of PLA Foci they contain.

Contributors

CLUET David	[email protected]
TERRONE Sophie	[email protected]

Publication

THE RNA HELICASE DDX17 CONTROLS THE TRANSCRIPTIONAL ACTIVITY OF REST AND THE EXPRESSION OF PRONEURAL MICRORNAS IN NEURONAL DIFFERENTIATION.

Marie-Pierre Lambert, Sophie Terrone, Guillaume Giraud, Clara Benoit-Pilven, David Cluet, Valérie Combaret, Franck Mortreux, Didier Auboeuf and Cyril F.Bourgeois

Nucleic Acids Res. 2018 Jun 21. doi: 10.1093/nar/gky545.

License

Copyright CNRS 2013

This software is a computer program whose purpose is to automatically detect Nuclei and Proximity Ligation Assay foci in human cells.

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 PLA 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
    • Original.jpg
    • Treated.jpg
  • [] macro
    • Close_Image.java
    • Command_Line.txt
    • Explorer.java
    • GUI.java
    • Main.java
    • Settings.txt
    • Treat_DAPI.java
    • Treat_RFP.java

Installation

The PLA macro requires 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 PLA 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/PLA folder. The shortcut Plugins/Macros/PLA will be added in the Menu bar.
6. Restart ImageJ to refresh the Menu bar.

1. Open FIJI.
2. Open the src folder of the PLA 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/PLA folder. The shortcut Plugins/Macros/PLA will be added in the Menu bar.
6. Restart FIJI to refresh the Menu bar.

Update

Follow the same instructions as for the installation process. As the settings for the detection of the nuclei and the PLA foci can be modified by the user, the Settings.txt file will not be affected. The Your setting file has been protected! message will indicate your analysis parameters have been preserved.