To begin place the healthy and degenerated Drosophila fly eye images in the same folder. Ensure that the files are appropriately named to identify the experimental groups and distinguish between males and females. Open the Ilastik software.
Click on Create New Project. Then from Other Workflows, select Cell Density Counting and select a location to save the project file. Sequentially, click on 1.
Input Data, Add New, and Add Separate Images to choose the standard image. Then click on 2. Feature Selection and select the features.
Choose more sigma values to increase sensitivity. Click on 3. Counting and set the foreground sigma value to 5.00.
Using the foreground tool, mark 50 or more individual ommatidia on the standard image. Click on 3. Counting again and use the background tool to mark the area outside of the ommatidia.
Draw green lines in a lattice shape around the ommatidia. Click on 4. Density Export and choose Export Image Settings to adjust image settings.
Then click on Output File Info and select format tiff for further analysis in Flynotyper. Click on 5. Batch Processing, Select Raw Data Files, and select all experimental fly photos to be analyzed.
The list of imported images to be analyzed is seen under Select Raw Data Files. Click Process All Files at the bottom of the 5. Batch Processing section.
After the software completes processing, check the folder containing the experimental fly eye images. Confirm that black images named, your sample name probabilities, are present. In ImageJ, open the Ilastik-generated tiff file.
Use the rectangle select tool to make a rectangle around the eye. Select Control X or Control C to crop the area and wait for a black box in the shape of the rectangle outline to appear. Sequentially, click File, New, and Internal Clipboard to open the cropped fly eye.
To save the cut fly eye as a JPEG, click on File, Save As, and JPEG. In ImageJ, click on Plugins, and Flynotyper. Under Genotypes, select Add Genotypes and open the folder containing the cut fly eye images.
Tick the Light Microscope and Vertically boxes. Then under Rank Ommatidia By, check Stability and Distance to the Center boxes. For the number of ranked ommatidia considered, set input as 200.
Click Run and wait for the results of the analysis to appear. Copy and paste the sample file and P-score into statistical software for further analysis. Drosophila eye images processed with Ilastik and analyzed under ring light showed clearer ommatidial patterns compared to those without ring light enhancing the qualitative assessment.
Flynotyper analysis indicated significantly higher phenotypic scores in the moderately and severely degenerated fly eyes compared to non-degenerated eyes. When Ilastik was not used, the P-scores between non-degenerated and moderately degenerated eyes were similar.
