In this study, we developed an electron microscopy method, called CryoChem, to optimally preserve the morphology of genetically labeled tissues. To test the quality of morphological preservation afforded by CryoChem, we performed Serial Block-Face Scanning Electron Microscopy (SBEM) on CryoChem-processed Drosophila antenna.
Brief description of sample preparation
After cryofixation by high-pressure freezing and freeze-substitution, cryofixed antennae were rehydrated gradually. Rehydrated samples was then subjected to DAB labeling reaction and stained using a high-contrast en bloc staining protocol. Next, the samples were dehydrated for resin infiltration and embedding, followed by imaging with X-ray microscopy and then SBEM.
Serial Block-face Scanning Electron Microscopy
The Drosophila antenna was imaged using a Gemini scanning electron microscope equipped with a 3View2XP and OnPoint backscatter detector. 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; pixel size was 6.5 nm; raster size was 12k x 9k; Z dimension was 1200 sections, and local gas injection was set to 85%.Volume was aligned using cross correlation, segmented, and visualized using IMOD.