TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
6	Grids float on the medium	Surface of grids is hydrophobic because of insufficient glow discharging Air bubbles are attached to the grid	Adjust the settings on the plasma cleaner (Step 5) Introduce grids into solutions slowly and vertically to avoid formation of bubbles (Step 6)
15, 31, 38	Carbon film is torn	Rough handling of the grids Forceps have been damaged Grids were allowed to dry out Carbon film was damaged during blotting	Handle grids gently Avoid using forceps with bent tips Do not aspirate grids to dryness (Steps 8 and 12; Box 1) Reduce compressed nitrogen gas PSI on Gatan CryoPlunge3 unit (Step 16)
	Too many cells on the grid	Seeding density is high Cells are over confluent	Use a lower initial seeding density (Step 13; Supplementary Table 1) Grow the cells for a shorter time (Step 14; Supplementary Table 1)
	Not enough cells on the grid	Low initial seeding density Cells are not adhering to the grid	Seed at a higher density (Step 13; Supplementary Table 1) Include growth factors or coat carbon with extracellular matrix proteins (Step 7; Box 1)
	Cells are too rounded	Low initial seeding density No extracellular matrix solutions applied to the grids Virus infection is too high	Seed at a higher density (Step 13; Supplementary Table 1) Coat carbon with extracellular matrix proteins (Step 7; Box 1) Lower MOI of virus (Step 15; Supplementary Table 2)
31, 38	Ice is too thick	Blotting conditions are inadequate Cells are too confluent	Use a longer blot time (Step 25) Remember to rotate blotters (Step 26) Reduce the seeding density (Step 14; Supplementary Table 1)
38	Ice is too thick on one half of the grid	Grid is not centered and held evenly in plunge device forceps	Make sure that tips of forceps do not extend past the rim of the grid (Step 21)
38, 48	Ice on the grid has a freckled appearance	Warming past the vitrification point (i.e., above –150 °C) has occurred	Check the temperature of the cryo-fLM stage (Step 28) and expedite grid transfers (Steps 27, 29, 35, and 37)
	Lots of cell debris in the ice	Cytotoxicity from virus infection or transfection Cells were damaged during blotting	Cryo-plunge at an earlier time point (Step 15; Supplementary Table 1) Add an extra or longer DPBS wash to remove debris (Step 22) Adjust the blot settings (Step 25)
	Cell membranes appear ruptured or blebby	Blotting conditions are too severe Grid became too dry before blotting Cells were exposed to low temperatures	Lessen the blot force (Step 25) Reduce exposure to air and evaporation (Steps 21–26) Reduce time spent out of the incubator Do not place culture dishes on a cold benchtop and avoid holding the grid near liquid nitrogen while loading the plunge rod (Step 24)
	Large amounts of solid ice particles on grid surface	Ice contamination from atmosphere	Do grid transfers in a humidity-controlled room (relative humidity <30%) Use Plexiglas shields or wear a facemask when transferring (Steps 27, 29, 35, and 37)
48	Gold fiducials aggregated in the ice	Initial solution of gold fiducials has precipitated	Vortex gold fiducial solution before use (Step 20)