cellDisplacement_DZNE

Scripts for assessment of neuronal and glial pathologies

Based on methods chapter: Wagner, Völkner, Schmied & Karl [in preparation].

Scripts & accepted datasets

Included in the package are the scripts for the workflow. Test files will be published on Zenodo.

cell-remodeling_nuclear-marker.ijm

Tissue ROI – aligned crop from a larger field of view

Single channel
Stack
TIFF

Test files: nuclear-marker

Detects in 3D the position of nuclei using a nuclear marker. Visualizes the location of the nuclei in 2D.

cell-remodeling_cytoplasmic-marker.ijm

Tissue ROI – aligned crop from a larger field of view

Dual channel
Stack
TIFF

Test files: cytoplasmic-marker

Performs an intensity based segmentation based on a cytoplasmatic marker. Gives a location of each pixel that is above the threshold.

Dependencies

Fiji - https://fiji.sc/

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. doi:10.1038/nmeth.2019

The script cell-remodeling_nuclear-marker.ijm needs the following update sites:

3D ImageJ Suite: http://sites.imagej.net/Tboudier/ Wiki: https://imagej.net/plugins/3d-imagej-suite/

J. Ollion, J. Cochennec, F. Loll, C. Escudé, T. Boudier. (2013) TANGO: A Generic Tool for High-throughput 3D Image Analysis for Studying Nuclear Organization. Bioinformatics 2013 Jul 15;29(14):1840-1.

Fiji > Help > Update

The ImageJ Updater window will pop up. Click Manage update sites and activate the 3D ImageJ Suite update site (3D ImageJ Suite: http://sites.imagej.net/Tboudier/ ) by making a tick mark next to it. Click Close and proceed with update by pressing Apply changes in the ImageJ Updater window and finally restart Fiji.

Workflow execution

Drag & Drop scripts into the Fiji toolbar. The scripts will be loaded into the Macro editor. Press Run for starting the script.

Analyze Nuclear Marker

Load cell-remodeling_nuclear-marker.ijm

Input directory: Folder that contains the input file(s).
Output directory: were to save the result files.
File suffix: specify the suffix of the input files.
Detection radius xy: xy radius for the 3D maxima finder.
Detection radius z: z radius for the 3D maxima finder (anisotropy in z).
Detection noise: noise setting for the 3D maxima finder – only local maxima are considered that are higher than this value.

Further documentation of the specific tools: 3D filter: https://imagej.net/plugins/3d-imagej-suite 3D Maxima finder: https://imagejdocu.tudor.lu/tutorial/plugins/3d_maxima_finder

Press OK to start the script.

Processing is performed in batch over all files with the correct File suffix in the Input directory.

The settings chosen will be saved with a time stamp as text file the Output directory (Settings_YYYY-MM-DD.txt). A Log file will open documenting the processing. This Log file will be also saved with a time stamp as text file in the Output directory (Log_YYYY-MM-DD.txt).

Results for each processed input image will be saved in the Output directory:

├── \<fileName\>_Detection.roi
├── \<fileName\>_Detection.tif
└── \<fileName\>_Results.xls

You can adjust the detection parameters by running the script and verifying the detections using the <fileName>_Detection.tif. Keep in mind that this is a 2D visualization of a 3D detection. Alternatively you can load the <fileName>_Detection.roi over the original 3D stack.

The coordinates as well as the height of the image is stored in <fileName>_Results.xls and can be used for further analysis.

Analyze Cytoplasmic Marker

Load cell-remodeling_cytoplasmic-marker.ijm

Input directory: Folder that contains the input file(s).
Output directory: were to save the result files.
File suffix: specify the suffix of the input files.
Name channel 1: name for result files.
Name channel 2: name for result files.
Median filter size: filter kernel size for median filter.
Rolling ball size: size for rolling ball background subtraction.
Threshold channel 1: automatic intensity based threshold.
Threshold channel 2: automatic intensity based threshold.