An accessible digital imaging workflow for multiplexed quantitative analysis of adult eye phenotypes in Drosophila melanogaster

The compound eye of Drosophila melanogaster has long been a model for studying genetics, development, neurodegeneration, and heterochromatin. Imaging and morphometry of adult Drosophila and other insects is hampered by the low throughput, narrow focal plane, and small image sensors typical of stereomicroscope cameras. When data collection is distributed among many individuals or extended time periods, these limitations are compounded by inter-operator variability in lighting, sample positioning, focus, and post-acquisition processing. To address these limitations we developed a method for multiplexed quantitative analysis of adult Drosophila melanogaster phenotypes. Efficient data collection and analysis of up to 60 adult flies in a single image with standardized conditions eliminates inter-operator variability and enables precise quantitative comparison of morphology. Semi-automated data analysis using ImageJ and R reduces image manipulations, facilitates reproducibility, and supports emerging automated segmentation methods, as well as a wide range of graphical and statistical tools. These methods also serve as a low-cost hands-on introduction to imaging, data visualization, and statistical analysis for students and trainees.