Cryo-FIB Milling of Yeast Cells

Cells were plunge-frozen and then cryo-FIB milled using the method of Medeiros et al., 2018 (link), as follows. Immediately before plunge-freezing, mid-log phase (OD₆₀₀~0.6) yeast cells were pelleted at 4000×g for 5 min. They were then resuspended in YPD media containing 3% (vol/vol) dimethyl sulfoxide as cryo-protectant to a final OD₆₀₀ of approximately 2.5. Four µL of the cells were subsequently deposited onto Quantifoil R2/4 200 mesh copper grids (Quantifoil Micro Tools GmbH, Jena, Germany), which were then manually blotted from the back with Whatman Grade 1 filter paper for approximately 3–5 s. The grids were then plunged into a 63/37 propane/ethane mixture (Tivol et al., 2008 (link)) using a Vitrobot Mark IV (humidity: 100%, temperature: 4°C). Cryo-FIB milling was performed on a Helios NanoLab 600 DualBeam (Thermo Fisher Scientific, TFS, Waltham, MA, USA) equipped with a Quorum PolarPrep 2000 transfer system (Quorum Technologies, Laughton, UK). Plunge-frozen yeast samples were coated with a layer of organometallic platinum using the in-chamber gas injection system and the cold deposition method (Hayles et al., 2007 (link)). Cryolamellae were then generated as follows: bulk material was first removed using the FIB at 30 kV 2.8 nA, followed by successive thinning of the cryolamellae at lower currents of 0.28 nA and 48 pA.