A Tape-Mounting Protocol for High-Resolution Z-Stack Imaging of Drosophila Compound Eyes

Here, we present a non-destructive mounting and imaging protocol for high-resolution phenotypic analysis of the Drosophila compound eye. Accurate phenotyping of the Drosophila compound eye is often limited by its convex geometry and mechanical fragility during handling. To address this, we detail a versatile, non-invasive imaging workflow that enables high-resolution visualization from multiple perspectives. The protocol utilizes a custom adhesive mounting strategy on an inverted-tape platform, securing flies by the body and wings while leaving the head completely untouched. Crucially, this flexible configuration supports distinct mounting orientations: a lateral position to capture the full convexity of individual eyes, and a frontal position to assess whole-head symmetry. This method also facilitates the arrangement of high-density arrays, allowing for the rapid processing of 20-30 samples per session. By integrating reflected-light stereomicroscopy with Z-stack acquisition and Extended Depth of Focus (EDF) reconstruction, the workflow generates uniformly sharp, quasi-3D composite images. This approach preserves natural pigmentation and produces high-fidelity, contrast-rich images suitable for quantitative organ-scale phenotypic analysis.