Focus Stacking Protocol for High-Resolution Insect Photography

Here, we present a protocol to acquire high resolution, extended depth of field images of insect specimens by photographic focus stacking using a modular digital imaging system. The method provides a standardized workflow linking equipment assembly, calibration, image acquisition, and post processing. Using a full frame mirrorless camera (61 MP) coupled to microscope objectives and synchronized strobe illumination, the protocol achieves pixel scales from 0.76 m-0.19 m and produces artifact free composites through sub-micron focus increments (0.2 m). The procedure can capture and process approximately 20 final images per week under routine laboratory conditions. Compared with existing stacking solutions, this low-cost hybrid setup (< 30% of the cost of commercial systems) maximizes accessibility while maintaining diffraction limited image quality. Representative applications include the production of color calibrated identification plates for taxonomy, biodiversity digitization, and outreach. The protocol's standardized structure facilitates reproducibility across laboratories and field stations, supporting large scale insect imaging campaigns in both resource limited and institutional environments.