Ice Contamination
Figure 2: Results of the Ice Contamination Survey to the question “On average, what percent of your cryo samples are ice contaminated?”
Te participants found different stages of the cryo-ET
workflow problematic. Forty-four percent of the participants found the transfer stages problematic (combined transfer from cryo-FLM to cryo-FIB/SEM and transfer between cryo-FIB/ SEM and cryo-TEM). Seventeen percent of the participants found the FIB milling stage problematic, while 11% of the par- ticipants found the cryo-FLM stage problematic (Figure 7). Te participants encountered a range of problems with their
Figure 1: Results of the Ice Contamination Survey to the question “What equip- ment do you use in your cryo-ET workflow?” presented as A) a pie chart and B) a Venn diagram. FLM, fluorescence light microscopy; TEM, transmission electron microscopy; FIB/SEM, focused ion beam/scanning electron microscope.
had some form of ice contamination and a weighted average of 33.2% of the lamellae had more than 50% ice-contaminated area ( Figure 3). A weighted average of 27.9% of lamellae were rendered useless due to ice contamination (Figure 4). We then asked the participants at what stage of the cryo-
ET workflow did they normally notice their samples were ice- contaminated. Tirty-six percent of the participants noticed ice contamination on their samples in the cryo-FIB/SEM, while 39% of the participants noticed it in the cryo-TEM, the last stage of the workflow (Figure 5). Ice contamination impacted the research of the cryo-EM
users quite evenly across the board. Te impacts were: decreased sample throughput, decreased sample yield, reduction in the quality of the tomograms, rendering their lamellae useless, and increased lamella preparation time (Figure 6).
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cryo-EM samples. Tey experienced ice contamination on the sample before FIB milling, “curtaining,” “leopard ice,” non-vitreous ice in their samples, ice crystals on top of the milled lamella, amor- phous ice layer on the lamella, and cracked lamellae (Figure 8).
Discussion Our results show that ice contamination is a profound issue
for a significant proportion of the cryo-EM users across the globe. Although the degree of damage varied among the participants, according to the weighted average, almost a third of the samples were rendered useless due to contamination and damage. It is unfortunate that the majority of users only found out samples were contaminated at the very last stage of the cryo-ET workflow, that is, in the TEM. Looking at the stages users find problematic, we can sort the ice contamination into 1) sample mounting, 2) sample transfer, 3) cryo-FLM, and 4) lamella preparation categories. Sample mounting and sample transfer-associated ice
contamination. Crystalline ice contamination can form on the sample during sample mounting and sample transfer due to moisture in the atmosphere, which was experienced by 18% of our survey participants (Figure 8). Currently, users typically perform AutoGrid assembly (known as C-clipping), shuttle or sample holder loading, and Autoloader cassette loading in
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