Versa 510 x ray microscope

Target expression of APEX2 in ORNs for SBEM was performed as follows^{46 (link)}. Briefly, transgenic Drosophila lines (10xUAS-myc-APEX2-Orco or 10xUAS-mCD8GF-APEX2) were generated to facilitate dendritic targeting of APEX2 ^{46 (link)}. Expression of APEX2 in select ORNs was driven by OrX-GAL4 drivers (Supplementary Data 1). Six- to eight-day-old female flies were cold anesthetized prior to the dissection of their antennae. The antennae were processed with the CryoChem method^{46 (link)}, which involves cryofixation by high-pressure freezing, freeze-substitution, rehydration, DAB labeling reaction, en bloc heavy metal staining, dehydration, and resin infiltration.
Microcomputed X-ray tomography was performed on resin-embedded specimens using a Versa 510 X-ray microscope (Zeiss) to determine DAB-labeled region of interest. The specimens were then mounted on aluminum pins with conductive silver epoxy (Ted Pella) and sputter coated with gold-palladium for SBEM imaging. The ab3, ab4, ac3II, at4 datasets were collected with a Gemini SEM (Zeiss) equipped with a 3View block-face unit (Gatan); the ab5 dataset was collected with a Merlin scanning electron microscope (Zeiss) equipped with a 3View2XP and OnPoint backscatter detector (Gatan). Parameters for SBEM image acquisition are listed in Supplementary Table 5.

Microcomputed tomography (microCT) was performed on resin-embedded specimens using a Versa 510 X-ray microscope (Zeiss). Flat-embedded specimens were glued to the end of an aluminum rod using cyanoacrylic glue. Imaging was performed with a 40X objective using a tube current of 40 kV and no source filter. Raw data consisted of 1601 projection images collected as the specimen was rotated 360 degrees. The voxel dimension of the final tomographic reconstruction was 0.4123 μm.