TABLE 1.

Troubleshooting table.

Problem	Cause	Solution	Example image
Beam not visible	Insufficient blotting, ice much too thick	Increase blotting time or number of blots or change the blotting paper	Figure 2b (TI)
Poor contrast	Insufficient blotting, ice too thick	Increase blotting time or number of blots or change the blotting paper
	Dense background from Buffer	Verify if the levels of sucrose, glycerol, etc. are below 5%
	Build-up of contamination in the column	Verify if anticontaminator is operational, desiccate or remove the film loaded into scope
Uneven or splotchy ice	Hydrophobic grid surface	Pre-clean grids; use fresh carbon and glow-discharged grids before applying the sample	Figure 2b (SP)
Excess contamination	Poor transfer or handling	Shorten the time the sample and cold tools are exposed to a wet environment	Figure 2b (BC)
	Ethane impurities	Use high-grade ethane; do not shake the ethane tank; when tank is close to becoming empty, change the tank	Figure 2a
Crystalline ice	Improper plunging	Verify that the grid is plunging deep and fast enough into the cryogen	Figure 2c (X), (V)
	Devitrification due to handling with warm tools	Precool tools and verify all contact surfaces are below −140 °C
	Poor thermal contact of grid with holder	Clean the sample boat or verify that the clip ring is tight
Freeze-dried sample	Severe heating, excess blotting (rare)	Verify that all contact surfaces are below −140 °C	Figure 2d
Degraded sample	Hostile substrate surface Properties	Use another batch of grids, clean grids and glow-discharge grids
	Elevated salt concentration due to on-grid evaporation	Use two-side blot; blot in a high-humidity environment