How The Ceres Clean Station and Vitri-lock Are Making Dry Rooms Obsolete

December 21, 2022 | News Type: Technology: , ,

Cryogenic electron tomography (cryo-ET) provides detailed 3D structural information of unique cellular landscapes making it a crucial technique for gaining a complete understanding of the disease state, drug interactions, and other biological processes. However, the sample preparation process associated with generating samples for cryo-ET is extremely difficult and time-consuming. Typical sample yields, even for skilled users, are only 10-15%. Streamlining the workflow will improve throughput and lead to better quality samples and therefore better tomographic data.

What are dry rooms used for?

The first step in making a cryo-ET sample involves rapidly freezing the samples on electron microscopy grids to vitrify them. This preserves the native structure while fixating them on a grid. From this moment on is where the real challenge begins: the samples must remain vitrified and free of crystalline ice throughout the rest of the workflow.

The frozen grids must be clipped with a ring frame to assemble them into an “autogrid” before the next step in the process. This is typically done in a liquid nitrogen bath to protect the samples from moisture in the air. Transfer of the grids from the liquid nitrogen bath the Cryo-TEM, Cryo-FLM or the Cryo-FIB/SEM must also be done under controlled conditions to ensure that the samples remain vitrified and minimize crystalline ice contamination.

In about 20% of facilities, low-humidity “dry” rooms are dedicated for cryo-EM/ET sample preparation to minimize exposure to moisture in the air [1]. Because low-humidity rooms can be expensive to build, maintain and operate, some labs utilize glove boxes in lieu of dry rooms. Glove boxes function to protect users against liquid nitrogen and provide a dry and enclosed environment for sample manipulation. Even with these measures in place, ice contamination remains a bottleneck on the overall sample yield.

Can a glove box be used for cryo-ET sample preparation?

Generic glove boxes are not designed with the cryo-ET sample preparation workflow in mind. Glove boxes do not generally have sample workstations that are required for manipulating, mounting, and clipping samples. During critical steps in which the vitrified sample must be transferred to and from the glove box, a transfer module is used, which is not usually integrated with the glovebox. This increases the potential for the sample to be exposed to the ambient environment, thus risking ice contamination. Several of the commercially available transfer modules can only maintain the samples in a vitrified state for a very limited time.

Delmic’s CERES Clean Station is a custom-designed glove box specifically for cryo-EM sample preparation. Users can prepare and transfer samples in an anhydrous environment (<1 ppm water), thus minimizing ice contamination. It is the first commercially available cryo-EM sample preparation station and has several key design elements that make it effective:

  • Air lock chamber allows for the loading and unloading of samples, supplies, and tools without perturbing the anhydrous environment inside the glovebox. Once the material is introduced to the load lock, the chamber is pumped down via a rotary vane vacuum pump and purged with nitrogen gas. A sliding metal table allows for the comfortable movement of samples into the enclosure. A touchscreen display allows for automatic operation.
  • Preparation station allows for manipulating, mounting, transferring, and clipping of samples. The station is mounted on an XY planar slide to allow for repositioning depending on the user’s preference. Within the station are modules for c-clipping, cassette loading, NanoCab loading, and shuttle loading. A liquid nitrogen feedthrough directly refills the module from a dewar outside the Clean Station. Additionally, a heated tool rack is available to remove moisture from collateral and prepare it for the next user.
  • Load lock for Delmic’s CERES Vitri-Lock or other cryo transfer module. The load lock is specifically used when transferring vitrified samples to and from the Clean Station to other instruments like the scanning electron microscope with focused ion beam (FIB/SEM) which is used for milling.
Delmic’s CERES Clean Station Dry Room
Purpose Anhydrous workstation forcryo-EM sample preparation and transfer A dedicated room with reducedhumidity
Cryogenic sample protection High Medium
Humidity level < 1% humidity 20-40% humidity on average
Installation + maintenance Easy installation and minimalmaintenance Complex installation, requiresextensive facility modification and constant maintenance
Mobility High Very low
Ability to leave samples protected while unattended Yes No
Support for vacuum transfer Yes Not without extreme modification

Which stage of the lamella preparation process is most problematic?

One of the most problematic steps of the cryo-ET lamella preparation process are the transfer steps. In a survey conducted by Delmic, over 40% of responses from 80 cryo-EM users across the world reported transfer steps as the most difficult steps in the workflow [1].

Why is sample transfer so challenging? This is because the delicate vitrified samples must be physically relocated for the next steps in the workflow: from vitrification to fluorescence imaging in an FLM then milling in the FIB/SEM and finally to the Cryo-TEM for imaging. During these steps, the sample essentially acts as a cold trap and will cause water molecules in the air to condense on its surface. In turn, this reduces the usable area for collecting cryo-ET data. Therefore, transfer devices need to provide both an anhydrous and sufficiently cold environment below the glass transition temperature of water.

Conventional transfer modules keep samples under low- to mid-vacuum and can only support a short window of “cold time”, making them logistically challenging to use. Unlike standard transfer modules, the CERES Vitri-Lock from Delmic keeps sensitive vitrified specimens under high vacuum (~10-5 mbar). This enables an anhydrous environment and cool times for up to 30 minutes, making it more user-friendly and reliable.

The Vitri-Lock consists of five key components:

  1. Transfer rod
  2. LN2 reservoir
  3. Vacuum sample chamber
  4. Valve
  5. Plug & play load lock adaptor for the Clean Station or the FIB/SEM

In summary, Delmic’s CERES Clean Station and Vitri-Lock work together seamlessly to minimize ice contamination and devitrification during critical steps of the cryo-ET sample preparation workflow. When used in combination with the CERES Ice Shield, which provides protection against ice contamination during milling in the FIB/SEM, researchers can improve their sample yield and acquire high-quality tomograms much more quickly.

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