I would like to be able to mill cells with FIB and then perform slice and view with an SEM. I hear milling is difficult without placing cells conductive surface even after coating cells with conductive materials e.g Gold. Could I seed cells onto a conductive coverslip or treat an SEM stub to make it able to support cell attachment?
Has anyone done this and can advise?
Thanks,
Joel
Isn't slicing with an ultramicrotome much easier and less damaging than the same with a FIB?
For Slice&View (FIB-SEM tomography) the cells should be embedded in resin (like for conventional TEM). A common way is to cultivate (or just adhere from a droplet) them on sapphire discs, then freeze with a high-pressure freezer (HPF), conduct freeze-substitution, and then embed in resin. We used such a preparation for FIB-SEM of sperm:
Article New Insights Into Sperm Ultrastructure Through Enhanced Scan...
Good luck!
It is possible to make coverslip into a "conductive" version by coating both sides of it with Indium Tin Oxide (ITO) and grounding it around the perimeter. Thin SiO2 windows with thicknesses suitable to serve as coverslip-like protective layer for FIB/SEM tomography are available from vendors of TEM membranes. Roughly the protocol would be as following: get TEM membrane with thin SiO2 window near the edge and coat both sides with ITO; grow or place cells on Si substrate and fix/prepare as needed; place membrane over the cells and bond to Si substrate with conductive glue or epoxy; do FIB tomo using TEM window as a protective layer instead of FIB Pt deposition. Because of added thickness of the SiO2 window this would be more practical for a plasma FIB. Best of luck!
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