Development of efficient fluorescence-guided FIB milling methodologies ("FIBucial" method)
In the Gonen lab, a lot of focus is put on solving structures of membrane protein crystals that require special growth solvants and conditions. Namely, several require the use of a tooth-paste like substance called lipidic cubic phase (LCP). When LCP matrix loaded with crystals is loaded on a grid, it looks like amorphous piles ressembling hills or mountains (sometimes several hundreds or nanometers high). It is impossible to see where the crystals are, and it can take a lot of trial and error to make a lamella right through the crystal.
We therefore use fluorescence guided plasma-FIB milling to target as best we can the crystals. While the X-Y correlation is quite straightforward and could be done by eye, the Z correlation, taking into account the different imaging perspectives of the SEM, FIB and our in-chamber fluorescence light microscope (iFLM), is impossible to do by eye.
When I arrived into the lab, the process of x,y,z correlation was not streamlined at all and the success rate was low. Additionally, the process took time, not because of milling time but correlation time. I thus setout to establish the best correlation pipeline to do the correlation on-the-fly, allowing for rapid targeting. Correlation methods often require landmarks, or fiducial markers that are electron-dense fluorescent spheres that can be spotted in all imaging modalities. Unfortunately, LCP is very hard to mix with aqueous fiducial marker mixes. I developed a method where the fiducial markers are directly “drilled” into the sample. I call them “FIBucials” and it allows quick x,y,z registration of the targets of interests allowing to focus the session time on the milling.
Although the streamlining of this method is still ongoing, it was used in two published manuscripts in Nature Communications (https://www.nature.com/articles/s41467-023-36733-4) and IUCrJ (https://journals.iucr.org/m/issues/2023/04/00/fq5021/index.html).
A focused paper on the FIBucial method is on its way.