Search
Cryo-electron tomography sample preparation workflow
Cryo-electron tomography (cryo-ET) enables 3D visualization of cells and tissues in near-native conditions, supporting high-resolution structural analysis across a wide range of biological samples, including macromolecular complexes, viruses, cells, organoids, and tissues.
Achieving high-quality cryo-ET data starts with optimized sample preparation, which directly impacts structural preservation, data fidelity, and final resolution.
Samples are vitrified using plunge-freezing for thin specimens or high-pressure freezing (HPF) for thicker, more complex samples. When samples exceed the thickness required for electron transparency, cryo-focused ion beam (cryo-FIB) or plasma-focused ion beam (PFIB) milling is used to produce thin lamellae suitable for high-resolution cryo-TEM imaging.
Cryo-ET sample preparation using plunge freezing
Cell preparation for cryo-electron tomography
For cellular cryo-ET workflows, samples are prepared using established cell culture techniques to ensure consistent growth and viability prior to vitrification.
Cells are seeded onto carbon-coated gold EM grids, which provide mechanical stability and support cell attachment. Grid surfaces can be functionalized with extracellular matrix components or adhesion-promoting agents to improve cell distribution and attachment.
Adherent cells typically require an incubation period of 1–7 days to allow for attachment, spreading, and morphological stabilization on the grid. Suspension cells can be applied directly to EM grids without this step.
Grids are then vitrified using the Thermo Scientific Vitrobot System to rapidly preserve native cellular ultrastructure for downstream cryo-ET analysis.
Locating the structure of interest can be difficult in the vast complexity of the natural cellular environment. However, through correlative light and electron microscopy (CLEM), various florescent labels can be used to tag cells.
For the cellular cryo-tomography workflow, the Thermo Scientific iFLM (Integrated Fluorescence Light Microscope) Correlative System combines light and electron microscopy into one system, eliminating extra sample transfer steps and enabling a streamlined cryo-correlative solution for cryo-tomography.
Cryo-lamella prepared with the Thermo Scientific™ Aquilos™ 2 Cryo-FIB. Image shows overlay of SEM image with fluorescence image obtained with iFLM Correlative System.
Cryo-ET sample vitrification by plunge freezing
Cryo-ET sample vitrification preserves cellular ultrastructure by rapidly freezing samples in a near-native, hydrated state.
Cells grown on carbon-coated gold EM grids are not stained but instead vitrified directly to maintain their native environment. During plunge freezing, rapid cooling converts liquid water into non-crystalline vitreous ice, preventing ice crystal formation that can damage cellular structures.
Vitrification is typically achieved using the Vitrobot System, which supports controlled blotting and rapid immersion of grids into liquid ethane at approximately −180 °C.
Successful vitrification depends on factors such as sample thickness and liquid film consistency, which must be optimized prior to freezing. After vitrification, grids are ready for transfer to cryo-focused ion beam (cryo-FIB) systems for lamella preparation.
.jpg "Thermo Scientific Vitrobot System")
Cryo-FIB lamella preparation and correlative imaging
Vitrified samples are thinned prior to 3D imaging in a cryo-TEM, such as the Thermo Scientific Krios Cryo-TEM or Thermo Scientific Glacios Cryo-TEM.
Sample thinning is a critical step in the cryo-ET workflow, as the electron beam can only penetrate specimens thin enough to transmit 200–300 keV electrons. Preparing electron-transparent lamellae enables high-quality tomographic data collection.
Cryo-focused ion beam (cryo-FIB) milling is commonly used to produce thin lamellae while preserving structural integrity. Compared to mechanical sectioning methods such as cryo-ultramicrotomy, cryo-FIB milling reduces artifacts associated with cutting at cryogenic temperatures, including compression.
Correlative light electron microscopy (CLEM) in cryo-ET
Correlative light-electron microscopy (CLEM) helps identify regions of interest within complex cellular samples, improving targeting accuracy during cryo-ET workflows.
Cryo-focused ion beam (cryo-FIB) systems enable precise thinning of vitrified samples at cryogenic temperatures (approximately −170°C), producing uniform lamellae for downstream imaging. These systems combine scanning electron microscopy (SEM) for visualization with a focused ion beam for controlled material removal.
In complex samples, locating specific structures can be challenging. CLEM addresses this by integrating fluorescence imaging to localize targets prior to or during lamella preparation. The Thermo Scientific iFLM Correlative System combines fluorescence and electron imaging within a single instrument, reducing transfer steps and enabling direct localization of fluorescent signals within lamellae to streamline targeting.
Thinning by milling
Following correlative localization, cryo-focused ion beam (cryo-FIB) milling is used to thin vitrified samples and to create lamellae containing the region of interest. Material is removed from above and below the target area to produce an electron-transparent lamella, typically 100–300 nm thick, suitable for cryo-TEM imaging. This process uses a focused gallium ion beam or plasma focused ion beam (PFIB) for controlled material removal.
This thinning step is essential for cryo-ET, as only sufficiently thin samples allow transmission of 200–300 keV electrons required for high-resolution tomographic imaging.
Automated cryo-ET lamella preparation
Automation streamlines lamella preparation by improving throughput and consistency across cryo-ET workflows.
Thermo Scientific AutoTEM Software enables automated, unattended lamella preparation with minimal user intervention, supporting efficient system utilization, including overnight runs. Following milling, lamellae can be re-imaged using the Thermo Scientific iFLM Correlative System to confirm target localization and support navigation during cryo-TEM imaging.
Prepared lamellae and associated metadata are then transferred to a Krios 5 Cryo-TEM or Glacios 3 Cryo-TEM for tomographic data acquisition.
Cryo-ET sample preparation using high pressure freezing
Cryo-electron tomography (cryo-ET) enables structural analysis of bulk biological specimens in near-native, fully hydrated conditions. This includes complex samples such as organoids, tissue biopsies, plant tissues, and multicellular model systems.
For these thicker specimens, conventional plunge freezing does not achieve sufficient vitrification. High-pressure freezing (HPF) is used instead to preserve ultrastructure in samples ranging from tens to hundreds of micrometers in thickness. During HPF, specimens are rapidly cooled under high pressure (approximately 2100 bar), enabling vitrification of samples up to 200 µm thick while maintaining structural integrity.
For downstream transmission electron microscopy (TEM) analysis, two primary sample preparation workflows are used: cryo-lift-out lamella preparation and Sandwich (or "waffle") method:
Cryo-lift-out lamella preparation
Cryo-lift-out enables site-specific extraction of regions of interest from high-pressure frozen bulk specimens, supporting targeted cryo-ET analysis of complex tissues.
Using a precision micromanipulator, such as the Thermo Scientific EasyLift NanoManipulator, integrated within a cryo-FIB-SEM system, a defined volume of material is milled and extracted under cryogenic conditions.
The extracted sample is attached to a dedicated grid type, such as a half-moon grid, and thinned into an electron-transparent lamella for cryo-TEM imaging.
This enables targeted structural analysis of protein complexes, macromolecular assemblies, and cellular architecture within native tissue environments.
Selected references:
- Tang, X. et al. Step-by-step procedure for an optimized serial lift-out cryo-focused ion beam milling technique in tissue analysis. Biophysics Reports (2025). DOI: https://dx.doi.org/10.52601/bpr.2025.250039
- Glynn, C., Smith, J., Grange, M., et al. A generalizable and targeted molecular biopsy approach for in situ cryogenic electron tomography of vitreous brain tissue. Cell Reports Methods (2025). DOI: https://doi.org/10.1016/j.crmeth.2025.101080
- Schiøtz, O.H. et.al. Serial Lift-Out: sampling the molecular anatomy of whole organisms. Nature Methods (2024). DOI: https://doi.org/10.1038/s41592-023-02113-5
- Nguyen, H. T. D., Perone, G., Klena, N., et al. Serialized on-grid lift-in sectioning for tomography (SOLIST) enables a biopsy at the nanoscale. Nature Methods (2024). DOI: https://doi.org/10.1038/s41592-024-02384-6
- Klumpe, S., Rigort, A., Plitzko, J. M., et al. Recent advances in gas injection system-free cryo-FIB lift-out transfer for cryo-electron tomography of multicellular organisms and tissues. Microscopy Today (2022). DOI: https://doi.org/10.1017/S1551929521001528
Sandwich (or “waffle”) method
The sandwich, or “waffle,” method is used to prepare thicker samples for cryo-ET by embedding them between carriers prior to high-pressure freezing.
In this approach, tens of micrometers of sample are loaded onto a cryo-EM grid and enclosed between two carriers. The assembly is then vitrified using high-pressure freezing (HPF).
After freezing, the grid is retrieved and processed using cryo-focused ion beam (cryo-FIB) workflows to produce electron-transparent lamellae suitable for cryo-ET imaging.
This method is particularly useful for samples that are difficult to orient or require additional support during preparation.
Selected references:
Kelley, K. et al. Waffle method: A general and flexible approach for FIB-milling small and anisotropically oriented samples. Nature Communications (2022). DOI: https://doi.org/10.1038/s41467-022-29501-3
Cryo-volume electron microscopy (vEM)
Cryo-volume electron microscopy (cryo-vEM) enables 3D imaging of vitrified samples by combining serial milling and imaging within a cryo-FIB-SEM workflow.
In cryo-FIB-based vEM, the specimen is sequentially milled using an ion beam, and each newly exposed surface is imaged using scanning electron microscopy and Thermo Scientific Auto Slice & View Software. The resulting image stack is reconstructed into a three-dimensional volume.
This approach supports visualization of structural organization across multicellular samples in near-native conditions. Volume imaging can also be used to identify regions of interest in unlabeled samples, enabling targeted lamella preparation for subsequent high-resolution cryo-ET analysis.
Resources
For Research Use Only. Not for use in diagnostic procedures.