We're interested in developing disease models for diseases like ALS, FTD, and multisystem proteinopathy to understand their molecular pathologies. One model we use is Drosophila Eye System. The regularity and symmetrical pattern of the eye can be used to evaluate the effect of introducing mutations and gene expression changes.
Our protocol addresses a gap in ways of quantifying rough eye phenotypes. Specifically, the gap created by manual ranking. Manual ranking is based on fusion, loss of Ommatidia, and bristle organization.
However, it can lead to bias. Therefore, combining Ilastik and Flynotyper creates for a more robust method for ranking abnormal eye phenotypes. Our protocol can be advantageous to manual ranking due to its robustness.
Even non-trained laboratory members can perform successful quantification, whereas more trained researcher is likely needed for manual ranking to avoid score to score differences between two different researchers. Therefore, this protocol can save time and resources. Our findings advance research because this protocol allows for more accurate quantification of rough eye phenotypes, especially weak alterations in eye morphology.
Due to bias when mainly ranking, finding Ilastik and Flynotyper will lead to more accurate scoring and therefore result in more accurate published book results. 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 one input data, add new, and add separate images to choose the standard image.
Then click on two feature selection and select the features. Choose more sigma values to increase sensitivity, click on three counting and set the foreground sigma value to 5.00. Using the foreground tool, mark 50 or more individual Ommatidium on the standard image.
Click on three 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 four 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 five 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 five 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 Image J open the Ilastik generated. tiff file. Use the rectangle select tool to make a rectangle around the eye.
Select ctrl plus X or ctrl plus 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 Image J 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 clear 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.
