Cryogenic electron tomography (cryo-ET) is a powerful technique that is used to obtain 3D volumes of macromolecular structures– such as cells, bacteria, or organelles – in their native environment at sub-nanometer resolution . A series of 2D transmission electron microscope (TEM) images are acquired over a range of tilts relative to the electron beam and post-processing of the image set is used to generate tomograms, essentially 3D images of the structure.
Cryo-ET specimens must be carefully vitrified so that ice crystals, which can damage the sample or cause artifacts, are not formed. Also, the specimens must be around 100 to 300 nm thick to be electron transparent. One of the most common methods of generating these thin sections, also known as lamella, is using a cryogenic focused ion beam equipped scanning electron microscope (cryo-FIB/SEM).
To better target the region of interest (ROI), the samples are labeled with a fluorescent dye and a cryogenic fluorescence light microscope (cryo-FLM) is used in conjunction with the cryo-FIB/SEM. During every step of the process, the sensitive sample must remain vitrified and free of ice contamination and mechanical damage to yield high-quality tomographic data.
The Cryo-ET bottleneck: low sample yield
The traditional cryo-ET sample preparation workflow is laborious and complex, resulting in only about 10-15% of usable lamella. Common issues seen in cryo-ET specimens include ice crystals obscuring the ROI, mechanical damage, missing the target ROI, and devitrification.
The workflow starts by mounting the biological samples on a EM grid. Plunge freezing or high pressure freezing of grids are different ways in which the biological material is vitrified in its native environment. Next, the grids are clipped in a liquid nitrogen bath to assemble “autogrid” cartridges, making them easier to handle.
The specimens are then loaded into a transfer module to bring them to a separate location for FLM imaging. Once the ROI has been identified by fluorescence microscopy, the sample is transferred from the cryo-FLM to the cryo-FIB/SEM for milling. Prior to imaging by TEM, the lamella may again be transferred to the cryo-FLM to confirm the presence of the ROI.
Each of the transfer steps can easily expose the sample to ice contamination and devitrification since common transfer devices have limited “cold times” and operate under low vacuum conditions. Additionally, cryo-FLM tools operate at atmospheric pressure which risks additional ice contamination. Furthermore, the lamella is coated with a thin layer of platinum to protect it from damage under the ion mill. Existing ice particles can impact the homogeneity of the platinum film resulting in image artifacts. Ice particles can also cause the delamination of the platinum layer thereby exposing the sample to damage and reducing the TEM data quality.
The low sample yield resulting from this cryo-ET sample preparation workflow leads to both wasted time and resources. This means that cryo-ET users will spend more time before they get useful 3D structural data.
Integrated cryo-CLEM for a simplified workflow
Integrating the FLM into the cryo-FIB/SEM can overcome the many challenges associated with conventional cryo-ET sample preparation workflows. Not only does this reduce the number of transfer and handling steps, thereby reducing the chance of ice contamination, but also eliminates the need for a separate cryo-FLM. An added advantages is that it also allows the user to perform guided lamella milling thus improving the ROI targeting. The simplified workflow improves sample yield while reducing the time spent by users in generating useful tomography data.
Delmic’s METEOR is the first commercially available integrated cryo-CLEM module that can be retrofitted to most cryo-FIB/SEMs. Its objective lens is parallel to the FIB beam, making it easy to switch between the in situ FLM image and FIB milling during guided milling. It is also compatible with a large range of objective options to fit the user’s needs, ranging from 10X to 100X and numerical aperture from 0.3 to 0.9. Single-bandpass or multi-bandpass filters are also available. Excellent YZ linkage is another key aspect that allows the sample to remain in focus while moving in the Y direction. This enables the visualization of large areas, tiling and stitching.
In an example shown here, Delmic’s METEOR was used to guide lamella milling of plunge frozen yeast cells where eGFP-tagged Ede1 was overexpressed to reveal endocytic protein condensates (END condensates) . Guided lamella milling allowed the user to confirm the region of interest through in situ FLM imaging throughout the entire milling process. The final lamella was confirmed to contain the END condensates and could be directly transferred to the cryo-TEM.
To further reduce ice contamination, Delmic’s CERES Ice Shield can be equipped on the cryo-FIB/SEM to prevent residual moisture from condensing onto the sample during milling.
 Milne, J.L.S., Borgnia, M.J., Bartesaghi, A., Tran, E.E.H., Earl, L.A., Schauder, D.M., Lengyel, J., Pierson, J., Patwardhan, A. and Subramaniam, S. (2013), Cryo-electron microscopy – a primer for the non-microscopist. FEBS J, 280: 28-45. https://doi.org/10.1111/febs.12078
 Smeets, M., Bieber, A., Capitanio, C., Schioetz, O., Van der Heijden, T., Effting, A., . . . Plitzko, J. (2021). Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ. Microscopy Today, 29(6), 20-25. doi:10.1017/S1551929521001280