The hippocampal neurons were cultured on sapphire disks. Details on how to prepare the hippocampal culture and the sapphire disk for this type of experiment can be found in the publication: Watanabe et al.; Flash-and-Freeze: Coordinating Optogenetic Stimulation with Rapid Freezing to Visualize Membrane Dynamics at Synapses with Millisecond Resolution; Front. Synaptic Neurosci. August 2016.
The 6 mm sapphire disk with the cultured neurons was placed in the recess of the middle plate (PCB). A spacer ring (100 µm thickness) was carefully placed on the sapphire disk so that cells are not damaged when the assembly with another sapphire disk is completed. To ensure stability of the assembly a cover ring was placed on top of the second sapphire. It is critical all parts to be non-conductive in this assembly (Fig.3).
Once the sample is frozen it is transferred to precooled acetone (-90 °C) in the automated freeze substitution unit Leica EM AFS2. The freeze-substitution was carried out in a mixture of 1% osmium tetroxide, 1% glutaraldehyde and 1% milliQ water in anhydrous acetone. Samples were kept 5-30 hours at -90 °C, 5 °C/hour to -20 °C, 12-16 hours at -20 °C, 10 °C/hour to 20 °C. Once the freeze-substitution was completed, specimens were embedded in Epon/Araldite resin Detailed description of the procedure can be found in Watanabe et al., 2016:Front. Synaptic Neurosci.
Fig.3: Assembly of two half cylinders and a middle plate /PCB for electrical stimulation in the Leica EM ICE