Fig. 3: Example of a prepared lamella containing an in celullo MgHEX-1 crystal and its electron diffraction (ED) characterization in a 200 kV cryo-TEM.

a EYFP-derived negative FL image of the targeted MgHEX-1 crystal, acquired around its Z-center under cryogenic conditions before the Ga⁺ FIB-milling procedure. Across more than three independent cell preparations, dozens of crystals were reliably identified on each frozen TEM grid. For ED analysis, the lamella was machined around the crystal center, had a thickness of ~250 nm, a width of ~4 µm, and was angled at 20° to the TEM grid plane. b 0.5 kV SEM image of the lamella prepared by cryo-FIB-milling; the inset shows an SEM image of the lamella with higher resolution. c 30 kV Ga⁺ FIB image of the lamella. (d) TEM image of the FIB-milled lamella containing a portion of the targeted MgHEX-1 crystal; the inset shows a TEM image of the lamella with higher resolution. e A 200 kV ED pattern obtained from a ~4.8 µm area of the lamella, defined by the parallel beam illumination and depicted by a blue circle in (d). The crystal volume impacting the ED signal (e) was estimated to be ~4.4 µm³ when excluding non-electron-transparent (black) parts in (d). f A 200 kV ED pattern collected from a 1 µm area of the crystal (crystal volume ~0.2 µm³), defined by a selected area aperture of the cryo-TEM and depicted by a red circle in (d). Both the ED signals were acquired at the same electron beam fluence of ~0.087 e⁻/Ų.