“…The generally preferred method is to add 5-20 nm gold nanoparticles to the sample prior to vitrification to act as fiducials during tilt-series alignment (Gruska et al, 2008;Dobro et al, 2017). The use of fiducials allows for accurate tilt-series alignment by correcting for non-rigid registration due to beam and stage-induced shifts as well as sample alterations (Karimi Nejadasl et al, 2013;Mastronarde and Held, 2017;Fernandez et al, 2018Fernandez et al, , 2019. However, adding fiducials to FIB-milled lamellae of eukaryotic cells has so far proven to be difficult.…”