Stemi 508 trinoc microscope

Adult flies were embedded in an agarose gel that was prepared in a 500 mL Erlenmeyer flask by mixing 2 g of UltraPure agarose (Invitrogen) with 100 mL of distilled water. The mixture was microwaved until bubbles were seen and the agarose was fully dissolved. Next, the Erlenmeyer flask with the dissolved agarose was transferred to a 60°C water bath (Thermo Scientific) to cool the agarose gel but maintaining it liquid. Flies were anesthetized with CO₂ and transferred to a 60 mm Petri dish (Falcon) filled approximately halfway with the liquid agarose gel. Wings and legs were submerged and the head was oriented with forceps such that one compound eye faced the microscope lens. Next, the Petri dish was placed on ice to allow the gel to solidify and then positioned under a Stemi 508 Trinoc microscope (model #4350649030, Zeiss); the eye was imaged using an Axiocam 208 HD/4k color camera (model #4265709000) set to auto exposure and auto white balance. Image processing was performed using Fiji (https://imagej.net/software/fiji/), Adobe Photoshop 2020, and Adobe Illustrator 2020 software.

Adult flies were immobilized by embedding them in an agarose gel. To prepare the agarose gel, we dissolved 2 g of UltraPure Agarose (Invitrogen, Waltham, MA, USA) in 100 mL distilled water. The solution was heated in a 500 mL Erlenmeyer flask for several minutes until bubbles were visible. The flask was then placed in a water bath (Thermo Scientific, Waltham, MA, USA) set to 58 °C to cool the solution down to 58 °C. Next, flies were anesthetized with carbon dioxide gas and transferred to a 60 mm petri dish (Corning, Corning, NY, USA) filled with about 10 mL of the liquid agarose gel. We then submerged the wings and legs using forceps and placed the petri dish on ice for the gel to solidify. After solidification of the gel, the petri dish was placed under a Stemi 508 Trinoc microscope (model #4350649030, Zeiss, White Plains, NY, USA), and the fly head was adjusted with the forceps such that one eye was oriented in parallel to the microscope lens. The petri dish was placed back on ice until imaging, which was performed with an Axiocam 208 HD/4k color camera (model #4265709000) set to auto exposure and auto white balance. Images were processed with Fiji (https://imagej.net/software/fiji/ accessed on 10 July 2022) [24 (link)], Adobe Photoshop 2020, and Adobe Illustrator 2020 software.