Cryogenic electron microscopy (cryo-EM) is an advanced high-resolution imaging technique used to gain insight into the smallest structures and interactions within biological materials in their native states. Specifically, cryogenic electron tomography (cryo-ET) is capable of generating 3D reconstructions, from thin lamella extracted from whole cells, at sub-nanometer resolution by collecting a series of 2D images at different tilts relative to the electron beam.
Knowledge gained from cryo-ET data can help scientists understand protein-protein interactions, molecular machinery, disease pathways and discover new drugs, for example. Despite this, the adoption of this technique within the life sciences community at large is hampered due to the long and laborious sample preparation workflow which often results in poor-quality lamellae. The current workflow requires several steps under conditions favorable for the formation of amorphous ice which impacts the resulting sample and data quality. Besides reducing the quality of 3D reconstuctions, ice contamination impedes productivity by requiring more time spent on generating quality samples that can be used.
In a recent survey conducted by Delmic, over 80 cryo-EM users were asked about how ice contamination impacts their research [1]. The result showed that ice contamination is a profound issue affecting a significant amount of cryo-EM microscopists. Not only is the quality of TEM data reduced due to ice contamination, but many samples are rendered useless.
How Does Ice Contamination Impact Researchers?
Survey results from cryo-EM users showed that ice contamination impacts them in multiple ways. About 40% of respondents reported that 20 to 40% of their samples were ice contaminated. Based on the weighted average of responses, about 43% of all samples contain ice contamination while nearly 30% are rendered useless due to ice contamination, representing a significant bottleneck on the overall workflow.
What are the Sources of Ice Contamination Throughout the Cryo-ET Workflow?
A typical cryo-ET workflow can be separated into four main segments: grid prep, FLM imaging, lamella milling in the cryo-FIB/SEM, and TEM imaging.
- Grid prep – Sample preparation and mounting can be a major source of ice contamination. In an effort to protect the samples from moisture in the air, sample manipulations are usually done in a liquid nitrogen-filled bench-top preparation station. Some facilities are also equipped with low-humidity rooms. The survey found that only about 20% of the facilities had access to low-humidity rooms. This can be attributed to the exorbitant cost and the logistical challenges in maintaining such rooms. But even these steps, LN2 environment in a low humidity room, are not 100% effective against ice contamination.
- FLM imaging – In order to identify and locate the regions of interest (ROI), the samples are usually labeled with a fluorescent marker and a cryo-fluorescence microscope (Cryo-FLM) is used to locate the ROI. In most cases, the FLM is a standalone instrument that operates at ambient pressure, raising the likelihood of ice contamination. The vitrified samples must be transferred from the preparation station to the cryo-FLM to map the ROIs for lamella milling which also increases the chance of ice contamination.
- Lamella milling in FIB/SEM – Once the ROI has be mapped out, the sample is transferred to the FIB/SEM then milled to generate lamellae. Ice contamination during this process can be significant with reported accumulation of amorphous ice of around 30 to 50 nm/hr. The amorphous layers decrease TEM image quality and limit the number of lamellae that can be prepared in one session.
Between each step in the process, the delicate, vitrified sample must be moved between different environments. Transfer modules are only maintained under low vacuum, therefore ice contamination is very likely to occur, even for skilled users, simply due to the need for multiple transfer steps.
Before milling, the specimen is coated by a thin layer of platinum to protect it from damage by the ion beam. Existing ice crystals on the surface can impact the layer coating uniformity and lead to image artifacts known as “curtaining”. Besides that, any ice particles that delaminate during milling will expose the sample to physical damage
When do Researchers Realize That Their Samples are Contaminated with Ice?
Unfortunately, ice contamination is generally not found until later in the workflow such as at the TEM or in the cryo-FIB/SEM. A large majority, about 75% of survey respondents, do not realize their samples are ice contaminated until they are at the milling or TEM imaging step. Thus, minimizing ice contamination at every step of the workflow would be immensely valuable to both individual users and core facilities that handle a high volume of projects.
Solutions for Minimizing Ice Contamination
Considering the many transfer steps and problems that can arise during each segment of the workflow, the Delmic CERES Ice Defence System effectively addresses these issues to minimize the sources of ice contamination. The complete solution is composed of three modules – the Clean Station, the Vitri-Lock, and the Ice Shield – which have been engineered with cryo-ET users in mind. The Delmic METEOR enables integrated fluorescence light microscopy (FLM) for guided lamella milling in existing cryo-FIB/SEMs. Below is a summary of how each tool works to improve cryo-ET sample yield.
| Workflow segment | Commercially available solution |
|---|---|
| Grid preparation | CERES Clean Station – gloved container with <1 ppm moisture, equipped with liquid nitrogen bath and manipulation tools |
| Sample transfer | CERES Vitri-Lock – transfer module that operates under high vacuum, allowing for longer cold time and minimal ice contamination |
| FLM imaging | METEOR – an integrated cryo-CLEM platform that can be retrofitted to cryo-FIB/SEMs. Allows for in situ FLM imaging and eliminates associated transfer steps leading to less ice contamination and better ROI targeting. |
| Lamella milling | CERES Ice Shield – cryo shutter that protects sample from amorphous ice growth during milling, automatically retracts for SEM imaging |
References
[1] Lau, K., Jonker, C., Liu, J., & Smeets, M. (2022). The Undesirable Effects and Impacts of Ice Contamination Experienced in the Cryo-Electron Tomography Workflow and Available Solutions. Microscopy Today, 30(3), 30-35. doi:10.1017/S1551929522000621
