Fe sem merlin compact vp

For serial sectioning, the blocks were trimmed with an Ultratrim diamond knife (Diatome) to a rectangle and a depth of 100 mm using an ultramicrotome (EM UC7, Leica Microsystems). Before sectioning, a carbon-coated Kapton tape (RMC Boeckeler) was plasma-treated using an ELMO glow discharge cleaning system (Agar Scientific) equipped with a homemade reel-to-reel motorized winder. Serial sections of 70 nm thickness were cut and picked up using an automated tape-collecting ultramicrotome (ATUMtome, RMC Boeckeler). After collecting the sections, the tape was mounted on 4-inch silicon wafers (University Wafer) with conductive double-sided adhesive carbon tape (Science Services). The wafers were then coated with a 5 nm carbon layer to ensure conductivity (EM ACE600, Leica Microsystems). Sections were imaged by a scanning electron microscope (FE-SEM Merlin compact VP, Carl Zeiss) equipped with the Atlas 5 Array Tomography software (Carl Zeiss). High-resolution serial images were taken with 5 nm-pixel resolution at 5 kV using a backscattered electron detector. The serial images were then concatenated, aligned and segmented using TrackEM (NIH). Segmented images were imported into Imaris software for visualization (Bitplane).

Adult flies were anaesthetized with CO₂, placed in 25% ethanol for 24 h at room temperature and dehydrated in a graded ethanol series for 3 days. Samples were dried from 100% ethanol with a critical point dryer (EM-CPD300, Leica Microsystems), gold-coated using a sputter coater (EM-ACE600, Leica Microsystems) and imaged at a magnification of 152X (FE-SEM Merlin compact VP, Carl Zeiss; operated at 3 kV).