Fly-QMA: Automated analysis of mosaic imaginal discs in Drosophila
(A,B) Conventional analysis of a mosaic eye imaginal disc. (A) Clones are identified by visual comparison of clonal marker fluorescence among nuclei. (B) Regions labeled homozygous mutant (−/−) or homozygous wildtype (+/+) for the clonal marker are compared with those labeled heterozygous wildtype (+/−) to assess whether reporter expression differs across clones. Fluorescence bleed-through is arbitrarily diagnosed. (C-H) Quantitative mosaic analysis. Panels depict a magnified view of the region enclosed by red rectangles in panels A and B. (C) Raw confocal image of the nuclear stain, clonal marker, and reporter of interest. (D) Segmentation identifies distinct nuclei. (E) Reporter expression is quantified by averaging the pixel intensities within each segment. Numbers reflect measured values. (F) Measurements may be corrected to mitigate fluorescence bleedthrough. (G) Individual nuclei are labeled homozygous mutant, heterozygous, or homozygous wildtype for the clonal marker. White arrows mark nuclei with ambiguous fluorescence levels. (H) Reporter levels are compared across clones to determine whether the perturbation affects reporter expression. Yellow region marks excluded clone borders. Comparison may exclude clone borders (yellow regions) and focus on a particular region of the image field (black arrows). In the eye imaginal disc, comparison is often limited to a narrow window near the MF (orange arrow).