Automated Tracking of Centrosomes

Publication

Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

Cluet D(1), Stébé PN(1), Riche S(1), Spichty M(1), Delattre M(1).

PLoS One. 2014 Apr 24;9(4):e93718. doi: 10.1371/journal.pone.0093718.eCollection 2014.

Author information:

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

Abstract: The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.

DOI: 10.1371/journal.pone.0093718

PMCID: PMC3998942

PMID: 24763198 [Indexed for MEDLINE]

Authors

CLUET David	david.cluet@ens-lyon.fr
SPICHTY Martin	spichty.martin@ens-lyon.fr
DELATTRE Marie	marie.delattre@ens-lyon.fr

License

Copyright CNRS 2013

This software is a computer program whose purpose is to automatically track centrosomes in DIC movies.

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 HERE

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 ACT macro requires ImageJ v1.47s or higher (Download).
• The .mov files require the Quicktime plugin for ImageJ (Download)

Files

• [] ACT
  • README.md
  • LICENSE.txt
  • [] doc
    • ACT_analysis.jpg
    • Logo_cnrs.jpg
    • Logo_ens.jpg
    • Logo_LBMC.jpg
  • [] src
    • Installation.ijm
    • Installation_FIJI.ijm
    • [] Macro
      • ACT_Motor_CommandLine.ijm
      • ACT_Table_CommandLine_creation.ijm
      • ACT.jpg
      • CMD_SUM.ijm

Installation procedure

By default your ImageJ program has no shortcut to the ACT macro. The objective of this installation procedure is to transfer automatically the ACT macro and all its required files within ImageJ/Macros sub-folder. Finally, for convenient usage, shortcuts will be automatically generated within the Plugins/Macros menu. If you have already a version of ACT, the installation procedure will overwrite it, making easy updating of your system.

The following procedure is described in our on-line installation tutorial video:

1. First launch the ImageJ program.
2. Open the folder you downloaded from our web site. It contains the current version of the ACT macro and the Installation.ijm file. Drag this file and drop it on ImageJ command bar (If you are currently working on FIJI please use the Installation_FIJI.ijm file). A new window will automatically pop up.
3. Use in the menu bar the Macros/Run Macro command.
4. The previous window disappears and License agreement window of the installation program is displayed. Check the license agreement box. Click on OK to proceed with the installation.
5. The installation is then performed in few seconds. At the end of the process the program inform you of the success of the installation.
6. Quit ImageJ to update the startup macro file (If you perform an updating of ACT, you can already use it without restarting ImageJ).
7. Once ImageJ is restarted you can see two new shortcuts in the Plugin/Macros menu:

• A.C.T.
• A.C.T. Command-Lines LAUNCHER

For further informations concerning the program please refer to the ACT_User_Guide_CeCILL_2014-02-28.pdf.