Aquilos 2 cryo fib sem dual beam microscope

Grids containing Y. entomophaga Ara-RoeA ∆YenLC, Ara-RoeA ∆HolA/Rz/Rz1 or Ara-RoeA ∆Rz/Rz1 strain cells were clipped in cryo-FIB-specific AutoGrids (Thermo Fisher) with alignment markers and a cut-out for milling at shallow angles. Clipped grids were transferred to an Aquilos 2 cryo-FIB/SEM dual beam microscope (Thermo Fisher). Lamella preparation was performed as previously described^{80 (link)}. In brief, after platinum sputter coating and deposition of metalloorganic platinum, clusters of bacterial cells were targeted for a 4-step milling procedure using decreasing ion beam currents from 0.5 nA to 50 pA. Milling angles of 6–10° relative to the grid were used. Lamellae were milled to a thickness range of 50–100 nm.

Grids were clipped into autogrids modified for FIB preparation^{62 (link)} and loaded into either an Aquilos or an upgraded Aquilos2 cryo-FIB/SEM dual-beam microscope (Thermofisher Scientific). Overview tile sets were recorded using MAPS software (Thermofisher Scientific) before being sputter coated with a thin layer of platinum. Good sites with parasites were identified for lamella preparation before the coincident point between the electron beam and the ion beam was determined for each point by stage tilt. Prior to milling, an organometallic platinum layer was deposited onto the grids using a GIS (gas-injection-system). Lamellae were milled manually until under 300 nm in a stepwise series of decreasing currents. Milling was performed at the lowest possible angles to increase lamella length in thin cells. Finally, polishing of all lamella was done at the end of the session as quickly as possible but always within 1.5 h to limit ice contamination from water deposition on the surface of the lamellae. Before removing the samples, the grids were sputter coated with a final thin layer of platinum. Grids were stored in liquid nitrogen for a maximum of 2 weeks before imaging in the TEM.