The Undesirable Effects and Impacts of Ice Contamination Experienced in the Cryo-Electron Tomography Workflow and Available Solutions
Katherine Lau,* Caspar Jonker, Jingyue Liu, and Marit Smeets Delmic Cryo B.V., Delſt, Te Netherlands
*
lau@delmic.com
Abstract: Cryo-electron tomography (cryo-ET) is a powerful tech- nique that can provide unprecedented insight into protein-protein interactions and molecular machinery in a near-native state. The adop- tion of cryo-ET by life science research groups is hampered by the challenges associated with cryo-ET sample preparation. The current sample preparation process has many steps at which ice contamina- tion may occur to negatively affect the final sample and data quality. A survey was conducted to better understand the effects and impact of ice contamination to the cryo-ET outcome. Over 80 cryo-electron microscopy users worldwide participated in our survey. The results are presented in this article. We furthermore discussed the currently available solutions that can alleviate the ice contamination problems to increase the sample yield and cryo-ET data output.
Keywords: cryo-electron microscopy, cryo-electron tomography, ice contamination, focused ion beam, subtomogram averaging
Introduction Cryo-electron tomography (cryo-ET) is a powerful micros-
copy technique that can reveal details in biomolecular inter- action in the near-native cellular environment at a nanometer scale [1]. Cryo-ET uses a transmission electron microscope (TEM) to acquire a series of 2D images at different tilt angles and 3D reconstruction to provide detailed structural informa- tion of biomolecules. To resolve the 3D structures of the protein complex at a high resolution, 1000–2000 copies of the protein complex need to be averaged [2]. Tis process is called subtomo- gram averaging. Due to the need for the high number of cop- ies, a high data throughput is desirable. While single particle analysis requires the protein of interest to be purified, in situ cryo-ET can offer novel insight into the biomolecular structure and protein-protein interaction within the cell. Cryo-ET has already been applied to the fields of structural
biology, microbiology, and virology [3,4]. Its ability to reveal information at a sub-organelle level has led to a better under- standing of cell biology, molecular physiology, and disease manifestations [4–8]. Tis kind of information can help in the development of targeted treatments and preventative measures for a variety of diseases [9]. Despite the powerful insight it can offer, the adoption of in situ cryo-ET is hampered by difficulty in obtaining good quality samples suitable for tomogram acqui- sition in the TEM. TEM imaging requires the sample thickness to be around
150–300 nm. Using a focused ion beam (FIB), typically com- bined with a scanning electron microscope (SEM), thin elec- tron transparent sections called lamellae can be prepared from cryogenic samples. A few major challenges in the cryo-ET sample preparation process exist. First, once flash-frozen, the samples need to remain vitrified throughout the cryo-ET work- flow. Second, crystalline and amorphous ice may form on the
30 doi:10.1017/S1551929522000621
samples during sample preparation and inter-device transfers, respectively, due to the moisture in the atmosphere. Tird, the molecules of interest may be absent from the thin lamella due to unsuccessful targeting. Last, but not least, an amorphous ice layer may form on the lamella inside the cryo-FIB/SEM due to the residual moisture inside the SEM chamber [10]. While ice particles on the lamella can obscure the region of
interest (ROI), amorphous ice layers can decrease the contrast of the TEM images and thereby data quality. To our knowl- edge, a large extensive survey on the ice contamination issue in cryo-electron microscopy (cryo-EM) has not been published. To further pinpoint the areas needing the most improvement to create the largest impact, we conducted a survey and invited cryo-EM users worldwide to participate.
Methods and Materials We designed a survey with 10 questions concerning ice
contamination issues experienced in the cryo-ET workflow. We invited cryo-EM users from around the world to fill out the survey. Te invitations were sent out by email, distributed through relevant cryo-EM network mailing lists, and posted on social media platforms including Linkedin, Twitter, and WeChat. Answers from participants who completed the sur- vey were compiled. Where appropriate, a weighted average was calculated.
Results Amongst the cryo-EM users who participated in the sur-
vey, the answers from 84 participants who completed the survey were compiled. Te participants came from 16 countries across Europe, North America, and Asia. We first surveyed the type of cryo-microscopy equipment
the participants used in their laboratories. About 35% percent of the participants used a cryo-TEM, 28% used a cryo-FIB/ SEM, 19% used a cryogenic fluorescence light microscope (FLM), and 18% used a low humidity room (Figure 1A). As shown in the Venn diagram (Figure 1B), not all cryo-TEM, cryo-FIB/SEM, and cryo-FLM owners used a low humidity room. Less than half (49%) of the cryo-FIB/SEM owners used a low humidity room. Next, we asked the participants to estimate the percent of
their cryo-EM samples that were ice-contaminated. Forty-one percent of the participants answered 20–40% of their samples had ice contamination (Figure 2). Te weighted average was 36.3%. Next, we sought to understand to what extent of the lamel-
lae used for tomogram acquisition were ice-contaminated. It was estimated that a weighted average of 43.2% of samples
www.microscopy-today.com • 2022 May
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72
