Table 3 ∣.

Troubleshooting table

Step	Problem	Possible reason	Solution
5	All the grids have floated.	Glow discharge is not sufficient to cause enough hydrophilicity, or grids are not used within a suitable time (30 min) after the glow discharge.	Glow discharging of grids should be done within 30 min before usage. Verify grow discharge condition.
12	The cells do not adhere properly.	Glow discharging is not sufficient. The cells need another type of extracellular matrix or higher density	See above. Optimize cell culture conditions.
31	Cryo-stage/Cryo-shield temperatures rise significantly above - 170 °C	N2 gas flow for cryo-shield and cryo-stage decreases significantly lower than needed.	Adjust the N2 gas flow at the source. If the same source of N2 gas is used for both the preparation station and cooling the cryo-stage, the prolonged use of N2 gas at the preparation station (e.g., for drying the preparation station) may sequester some N2 gas and reduce its flow rate into the cryo-stage and cryo-shield. A leak of cold N2 gas inside/along the heat exchanger. Check all the tubing connections. Unusual frost observed in the heat exchanger often indicates a location of cold N2 gas leak.
57 & 59	Ice contamination on top of the sample before milling	Contaminated liquid N2 during plunge freezing, clipping, and sample loading If the samples are observed by cryo-fluorescence microscopy, some degree of contamination should be accepted. Vacuum issues during the transfer	Reduce room humidity Dry liquid N2 dewars before filling with fresh liquid N2 Filter liquid N2 with coffee filters Wear a mask to prevent contamination from the breath Carefully dry tools before re-use Check and clean valves and O-rings
57 & 59	Many broken EM grid meshes (Fig. 9a)	Damage during seeding cells on grids, plunge freezing or clipping	Evaporate a thin carbon layer on top of Quantifoil grids using carbon evaporator to make the substrate stronger Consider Quantifoil grids with SiO2 film known to provide more robust and stiffer support89. Manipulate grids more gently. Careful and gentle clipping, e.g., approach the EM grid in the FIB-AutoGrid with the C-ring Insertion Tool vertically; do not press the C-ring Insertion Tool vigorously onto the FIB-AutoGrid while pushing the C-ring down.
57 & 59	No cells on the EM grid	Insufficient glow discharging of grids Cell concentration too low	Use glow discharged grids within 30 min Optimize parameters for glow discharge system Optimize cell concentration during seeding on the grids
64	When SEM and FIB do not show the spot on the grids even at the eucentric height.	The microscope is not well-aligned.	Reset beam shifts both in SEM and FIB. Use beam shift to adjust digitally. If still not possible, it is highly likely the FIB column position/angle needs to be adjusted mechanically.
70	Identification of cells on grids hindered after Pt Deposition	Thick Pt deposition. Check the thickness by the shape of the shadow of the protruding features. Pt deposition occurs at a slight angle, thus leaving behind a shadow for protruding features.	Lower the Pt deposition time Changing the grid-needle distance. Changing the temperature of the GIS is also, not recommended.
66-73	FIB image moves	GIS Pt layer is not yet cured. Too thick ice on the entire sample Ice contamination along the focused ion beam path. The grid bar is along the FIB beam path. If temporal, it could be due to the change of stage temperature	Use FIB to bake (Fig. 8b) Prepare a new sample. Increase the milling angle, if possible. Select a different area for milling. Increase milling angle Check stage temperature.
66-73	Milling takes an extremely long time	Platinum layer is too thick Milling the grid bar	Reduce the thickness of the platinum layer by reducing the application time and/or increasing the GIS sample-to-needle distance Adjust milling position. Get familiar with how grid bars look and stop milling
66-73	Lamella bending/moving while milling	Charging is likely causing the beam-induced movement of the lamella Shrinkage of the EM grid support film at cryogenic temperatures	Apply platinum layer Increase platinum layer thickness Carefully supervise milling process and compensate sample movement with ion beam shifts. Make micro-expansion joints84
66-73	Lamella breaks while milling	Lamella too thin Beam-induced movement Shrinkage of the EM grid support film at cryogenic temperatures	Make a thicker lamella See “Lamella moves while milling” Make micro-expansion joints84
99	Vacuum pressure failure during the microscope warm-up	As water molecules frozen onto cold surfaces (e.g., cryo-shield) releases upon warming up, the pressure rises	Immediately activate pumping of the Microscope chamber.
104-105	Ice contamination on top of the milled lamellae	Contaminated grids Contaminated liquid N2 during transfers Vacuum issues during transfer	Reduce contamination during grid preparation See “Ice contamination on top of the sample before milling” Check and clean valves and O-rings
104-105	Curtains	No or too thin platinum layer Crystalline ice on the surface before Pt coating The FIB current too high during milling The materials of significantly different density are present in the biological materials	Increase the thickness of the platinum layer by increasing the application time and/or reducing the GIS sample-to-needle distance Minimize ice contamination Use lower FIB current to make smoother lamellae Make sure to complete material milling at each step before proceeding to the next milling.
104-105	The back part of the lamella is significantly thicker than front part (close to Pt layer).		Make sure to complete material milling at each step before proceeding to the next milling. Use different angles (plus/minus 0.5 °) for final milling step83
104-105	Crystalline ice in the samples	Sample too thick	Increase blotting time Decrease cell concentration on the grid. If cells are too large for plunge freezing, this protocol might not suitable
104-105	“Leopard ice” on top of the lamellae	Shuttle/Stage temperature issue	Monitor stage top and shuttle temperature with thermocouples. Check the N2 flow of the heat exchanger. Inspect and clean surfaces of the upper and lower part of the cryo-stage and cryo-bearing. Retrieve the prepared EM grid from the microscope chamber as soon as possible after milling
106	Sample not aligned with milling direction 90° in regard to the tilt axis	Grids are not well-aligned in the shuttle for dual beam microscopy and/or in Titan cassette	Make sure to mark AutoGrid properly and orient them properly in dual beam and TEM.
106	Beam-induced motion while acquiring tilt series	Charging The lamella is too thin The lamella has cracks	Consider coating lamella with Pt sputter coating Create thicker samples Gentle grid manipulation. See “Lamella bending/moving during milling” “Lamella breaks during milling”
107	Difficulties aligning tilt series	Lamellae are too thick to render good contrast The appearance of crystalline ice inside of samples Unstable Record/Tracking areas	Make thinner samples. See below “Invisible amorphous ice layer on the top and/or the bottom of the reconstructed tomogram” Decrease pixel size Need to increase total electron dose See “Crystalline ice in the samples” Choose suitable area for tilt series acquisition. Gentle grid manipulation. See “Lamella bending/moving while milling” “Lamella breaks while milling”
106-107	Invisible amorphous ice layer on the top and/or the bottom of the reconstructed tomogram. The existing of this can be only confirmed by the observation that contaminated crystalline ice particles are not directly located on the biological layer.	Redeposition during milling Contamination in Autoloader in Titan Krios	Perform final fine milling just before retrieval of samples as mentioned in step 71. Perform “Cryo-cycle”