In 2018, the Su and Ellisman group developed a novel electron microscopy technique called CryoChem, designed to achieve optimal morphological preservation of genetically labeled tissues. To evaluate the preservation quality provided by CryoChem, we performed Serial Block-Face Scanning Electron Microscopy (SBEM) on CryoChem-processed Drosophila antenna.
Sample Preparation and Imaging Overview:
Following the CryoChem protocol (Tsang et al., 2018), a Drosophila melanogaster antenna expressing membrane-tethered APEX2 in ab4A ORNs (Or7a GAL4 driver) was prepared for high-resolution volume EM. The antenna was isolated, cryofixed using high-pressure freezing, and processed through freeze-substitution, rehydration, en bloc heavy metal staining, dehydration, and Durcupan resin infiltration.
Microcomputed X-ray tomography was used to orient the sample, which was subsequently mounted on an aluminum pin with conductive epoxy and sputter-coated with gold-palladium. Imaging was performed using a Gemini SEM 300 (Zeiss) equipped with a Gatan 3View2XP system and an OnPoint backscatter detector. The SBEM imaging parameters included 2.5 kV accelerating voltage, 30-μm aperture, 1 μs dwell time, 5 nm per pixel resolution, and 40 nm Z-step size. To minimize charging artifacts, focal charge compensation with nitrogen gas was applied.
