In this study, we developed an electron microscopy method, called CryoChem, to optimally preserve the morphology of genetically labeled tissues. This method also allows us to perform 3D correlated light and electron microscopy (CLEM) in the well-preserved tissue. This dataset contains the SBEM volume of the CLEM experiment in a mouse brain expressing tdTomato in corticotropin releasing factor-expressing neurons.
Brief description of sample preparation
After cryofixation by high-pressure freezing and freeze-substitution, cryofixedmouse brain slices were rehydrated gradually. Rehydrated samples were then imaged with confocal microscopy to capture the DRAQ5 and tdTomato signals. Next, the samples were stained using a high-contrast en bloc staining protocol. Then 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 mouse brain slice wasimaged with a Merlin scanning electron microscope equipped with a 3View2XP and OnPoint backscatter detector. The volume was collected at 2 kV, with 6.8 nm pixels; 70 nm Z steps and 15% local gas injection. The raster size was 10k x 15k and the Z dimension was 659 sections. The volume was aligned using cross correlation and visualized using IMOD.