Our images taken with the Nikon Spinning Disk confocal microscope are classified according to the following rationale: Cell type > stress > Date of experiment > microscopy field
Tree definition:
-Cell type: corresponds to the cell line used
-Stress: corresponds to stress applied to cells: -Nostress (basal condition) -SD: serum deprivation (elongation of mitochondria) -UVB500: UVB irradiation at 500mJ/cm^-2 (fission of mitochondria)
-Date: Date the image was taken
-Microscopy field: Images taken at a given position
In each field folder, there are 4 images in .tiff format corresponding to images taken at the same position on the channels:
- Green (CellMask Green): cellular contour
- Blue (Hoechst): nucleus
- Red (MitoTracker Red): mitochondria
- Merge: superposition of the three previous images.
Images can be viewed using Fiji ImageJ software --> https://fiji.sc/
MitoTouch features:
All files ending in "MERGE.tif" that are included in a folder named "Champ - Copie (X) " will be processed in the selectionned tree directory.
It is higly recommended to test MITOTOUCH parameters on 1 image of the the selectionned tree directory before processing analysis.
Load test image: Allows you to load and display a test image, in order to refine the selection parameters
Test: Allows you to reload the test image after changing the parameters
Nuclei threshold : Ability of the software to detect the nuclei of the cells, which will be surrounded in yellow. Increasing it means that the selection will be stricter. A value that is too low can lead to selecting elements that are not nuclei, a value that is too high can lead to not selecting nuclei.
Cell threshold : Ability of the software to detect the contour of the cells, which will be surrounded in pink. Increasing it means that the selection will be stricter. A value that is too low can lead to selecting elements that are not part of the cells, a value that is too high can lead to not selecting certain cells.
Cell erode : Determines the contour accuracy of the cell contour selection. A higher value will have finer contours
Filter border cell : Allows to select not complete cells that must be present in the image for the software to select it. A value of 0% will select all partial cells, while a value of 100% will only select cells completely in the image.
Export mito based data : Check this option if you want to have parameters for each mitochondria instead of a mean of mitochondrial parameters per cell.
Display : Allows to check the selection on the test image in order to check the accuracy of the parameters
Original: Displays the original image Nuclei: Displays the selected nuclei Cell: Displays the contour of the selected cells Mito : Displays mitochondria Skeleton: Displays the skeleton of the mitochondria Final: Displays all elements
Load test image: Allows you to load and display a test image, in order to refine the selection parameters
Test: Allows you to reload the test image after changing the parameters
Run : Allows you to perform processing under the selected folder. All files ending in "MERGE.tif" that are included in a folder named "Champ - Copie (X) " will be selected for processing CAUTION, this also includes the entire descending file tree of the selected folder.
abdel.aouacheria (at) umontpellier.fr & victor.racine (at) quantacell.com
Parallel : Distributes the operation over the different cores of the computer to increase processing speed CAUTION, due to the power required, a minimum of 16 GB of RAM is recommended.
At the end of the analysis a "result" folder in format will be created in the selectionned tree directory. It will be composed of all the images processed by MITOTOUCH and a .csv file containing all the numerical results.