Image Analysis using FIJI / ImageJ

This tutorial will aid the user in analyzing the folder of images obtained from imaging zebrafish larvae in 96-well plates.

It is assumed that the user will have these images stored in the computer that they will use to perform the analysis. Alternatively, the images can be stored on the R-drive or in a portable hard drive.

Materials

FIJI/ImageJ

Zebrafish Macro (Supplementary File 2: 41598_2022_10133_MOESM2_ESM)

Installing FIJI / ImageJ

1. Download the version of FIJI that matches your computer (e.g. if you are using a mac, download the macOS version) by clicking on the link from the list of materials.

2. Extract the downloaded file (right click on the file -> extract here) and click on the launcher to open FIJI/ImageJ.

   Open the installation folder and launch FIJI/ImageJ

   

3. Click on the ‘Plugins’ tab and then click on ‘Install…’ to install the zebrafish_macro26rc062019.ijm if you haven’t installed it before.

   Click the Plugins tab

   

   Install the macro

   

4. Click ‘Open’ to install the macro.

5. Close FIJI/ImageJ.

Analyzing behavioral images using ImageJ/Fiji

1. Open FIJI/ImageJ.

2. Click on the ‘Plugins’ tab and then scroll down until you find the zebrafish_macro26rc062019 macro.

3. Click on ‘zebrafish_macro26rc062019’ and enter the following settings:

   Analysis settings for four 96-well plates

   

4. You can change the name of the ‘Results’ file in the first option if needed. Click ‘Ok’ once you’re done.

5. Open the folder where your images are and find the number of the first image in green. For example:

   Open the folder and look for the image

   The number of my first image in green is 801.

6. Put the number of your first image in green in the first box.

   Enter the image number

   

7. Repeat the same process for your first image in blue.

8. Find the first image in red that happened after your blue images (not before). For example:

   First image in red after blue images

   

9. Put this number in the ‘First image in yellow’ box. Then, click ‘Ok’.

10. Open the first image in your behavioral images folder by clicking on the ‘File’ tab, then ‘Open’.

    Open the first image

    

11. Click ‘Ok’. Your image will have a yellow rectangle on the top left corner. Move this rectangle so that it fits around the first row of your first plate.

    Draw a rectangle around the first plate's first row

    

12. Once you’re done fitting the yellow rectangle, click ‘Ok’.

13. Once again, take the yellow rectangle and fit it around the second row of your first plate and click ‘Ok’.

    Draw a rectangle around the first plate's second row

    

14. Continue to do this with all the rows on all the plates, even if some of your plates are empty. You will start with the rows in plate 1 and then move down to plate 2, up to plate 3, and down to plate 4.

15. Once you’re done, the software will start analyzing the images. When it’s done, the Results file will be in the folder with your behavioral images.