Focused Ion Beam Scanning Electron Microscopy for 3D Reconstruction of Synaptically Linked Neurons

Take a fixed section of mouse cortical brain tissue exhibiting neuronal axons and dendrites. The dendrites possess protrusions, known as dendritic spines, which form synapses with axons.

Incubate the tissue with osmium-ferrocyanide, which binds to lipids, followed by another osmium application, which enhances structural contrast for imaging.

Stain with a heavy metal compound that binds to macromolecules and enhances contrast.

Dehydrate using increasing alcohol concentrations for resin embedding.

Embed the tissue in resin, mount it on a holder, and coat it with gold to optimize imaging quality.

Use focused ion beam scanning electron microscopy for three-dimensional imaging.

Employ an ion beam to remove a thin layer of resin-embedded tissue, exposing an imaging surface.

Focus an electron beam on the exposed surface and detect the backscattered electrons to generate an image.   
 
Perform serial slicing and imaging and reconstruct a three-dimensional image, revealing a synaptically linked cortical neuronal network.