Conductive silver epoxy

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.

Following microcomputed tomography to confirm proper orientation of region of interest, specimens were mounted on aluminum pins with conductive silver epoxy (Ted Pella, Redding, CA). The specimens were trimmed to remove excess resin above ROI and to remove silver epoxy from sides of specimen. The specimens were sputter coated with gold-palladium and then imaged using a Gemini scanning electron microscope (Zeiss) equipped with a 3View2XP and OnPoint backscatter detector (Gatan). Images were acquired at 2.5 kV accelerating voltage with a 30 μm condenser aperture and 1 μsec dwell time; Z step size was 50 nm; raster size was 12k × 9k and Z dimension was 1200 sections. Volumes were either collected in variable pressure mode with a chamber pressure of 30 Pa and a pixel size of 3.8 nm (Figure 2—video 1 and Figure 3D) or using local gas injection (Deerinck et al., 2018 (link)) set to 85% and a pixel size of 6.5 nm (Figures 2A, B and 3C). Volumes were aligned using cross correlation, segmented, and visualized using IMOD (Kremer et al., 1996 (link)).