METEOR Target: New workflows for various samples developed with and by leading cryo-ET labs

This developer story highlights the development and uses of METEOR Target for:

• Thick High-Pressure Frozen (HPF) samples for waffle milling or lift-out, 
• Automated milling for cells-on-grid with many targets, 
• and the SuperZ method for tiny, sparse targets in cells-on-grid that need high-precision Z localization.

These techniques were presented by users and developers during the METEOR Target Webinar. You can watch the full replay here: https://watch.getcontrast.io/register/delmic-meteor-target-webinar 

In cryo-ET, the difference between a usable tomogram and wasted FIB-SEM time often comes down to one question: did your target end up inside the lamella? Over the past few years integrated fluorescence microscopy with METEOR for cryo lamella milling has helped many labs to capture their target proteins in their lamellae [1, 2, 3]. But as the field is moving forward to more complex samples [4, 5], the demands for integrated fluorescence microscopy are changing as well. Therefore, together with leading labs in the cryo-ET field, we further developed the METEOR workflows to meet the future needs of the cryo-ET field. The result: METEOR Target. METEOR Target improves the lamellae preparation workflows for high-pressure frozen samples that need accurate 3D targeting, cells-on-grid with plenty of targets that benefit from automated milling, and cells-on-grid with very sparse and small (<400nm) targets that need precise z-localization (3D). Read on to learn directly from the developers themselves how you can use these techniques for your samples.

High-pressure frozen samples: FIB-view fluorescence microscopy

Thick high-pressure frozen (waffle) samples present a major targeting challenge. Standard top-view fluorescence often turns puncta into blurred columns, and z-localization becomes difficult because the sample surface lacks reference points or fiducial beads.

Therefore, Cristina Capitanio and Anna Bieber (MPI of Biochemistry) developed FIB-view fluorescence microscopy [6] to help you capture your target in your lamellae, even in HPF samples. Once your trenches are milled, this technique enables you to image your targets with the fluorescence microscope from the same perspective as the focused ion beam. This approach makes it possible to:

• Improve the resolution for 3D targeting by changing the imaging angle
• Easily correlate fluorescence and ion beam images in 2D for targeted milling
• Capture your target inside the final lamella

Watch how you can use this technique as it is explained by one of the developers, Cristina Capitanio:

Did you know that you can also apply this technique for cells-on-grid?!

Cells-on-grid with plenty of targets: All-in-one automated milling software Odemis Advanced

For cells-on-grid with abundant targets, you want to focus your workflow on speed, consistently keeping the target inside the lamella, and reducing manual effort. We noticed that people often used a multitude of software suites to perform automated milling, SEM overview imaging, FM-imaging [7], and 3D correlation [8]. And next to this requiring a lot of time to import and export data and images from one software suite to another, it also takes a lot of time to get people trained on all the different software suites. This was especially noticeable at large multi-user facilities like SLAC National Accelerator Laboratory. Therefore we partnered with SLAC to beta test our new solution: Odemis Advanced.

Odemis Advanced, part of the METEOR Target release, makes it possible to easily and accurately use FM data to guide automated milling and all the steps in between. This streamlined workflow supports:

• SEM- & FM overview imaging in the same software
• Automated fluorescence overlay directly on the SEM overview (2D correlation)
• Integrated 3D correlation (FM z-stack and FIB images) for accurate z-targeting
• Automatic milling of multiple sites overnight

Lydia Joubert from SLAC started using this and this is what she likes about it:

This setup was especially designed for samples with many targets in each cell. The system supports fast preparation and reduces repeated switching between computers and software tools.

Join Lydia Joubert for a step-by-step tour of the software in these videos:

Finding your milling sites:

3D correlation and placing milling patterns:

Automated milling & Fluorescence check:

Metadata & TEM tomogram:

Sparse small targets: SuperZ for precise 3D targeting

Sparse targets present a different problem. When the structure of interest is small, faint, and sparse, standard targeting can miss your target and fail to capture it in a lamella. SuperZ addresses this. It improves z-localization through a cylindrical lens system while still using a high NA objective to capture all the necessary light of even the faintest of targets [9]. We validated the hardware functionality at MPI of Molecular Physiology, co-developed the workflow in the lab of Sebastian Tacke and now they are using it to keep their targets inside the final lamella.

The workflow is as follows:

• Rough mill a flat top surface of the lamella
• Use the automated SuperZ hardware to determine the z-position of small targets with high accuracy & precision beyond the limits of standard fluorescence microscopy. 
• Perform 3D correlation based on fiducial beads visible in both the FM z-stack and the FIB image.
• Let the software correct for refractive index mismatch
• Fine mill and polish your lamellae based on the SuperZ data and retain your very small and sparse structures within the lamella.

Check out Sebastian Tacke’s results in this video:

Conclusion

These three examples show how METEOR Target enables integrated fluorescence microscopy for different sample types with different needs. Together, these techniques point toward a more efficient cryo-ET workflow with less manual transfer of data, faster targeting, and stronger confidence that the target remains inside the lamella.

Want the full detail, including each developer walking through their own workflow? Watch the webinar at: https://watch.getcontrast.io/register/delmic-meteor-target-webinar

References

1. ESCRT proteins organise stress-induced organelle contact between endoplasmic reticulum and Golgi  Pajonk, O. et al (2026) BioRxiv
2. In situ chromatin dynamics and HIV-1 nuclear trafficking Hou, Z. et al (2026) BioRxiv
3. A generalizable and targeted molecular biopsy approach for in situ cryogenic electron tomography of vitreous brain tissue Glynn, C. et al (2025) Cell. Rep Methods
4. Streamlined Montage Cryo-Electron Tomography for Exploring the Ultrastructure of Cells and Tissues Hylton, R. et al (2025) BioRXiV 
5. Serial lift-out – sampling the molecular anatomy of whole organisms Schiotz, O. H. et al (2023) Nature Methods
6. Making plant tissue accessible for cryo-electron tomography, Pöge, M. el al (2025), eLife
7. Odemis microscopy software github by Delmic B.V. https://github.com/delmic/odemis
8. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy Arnold, J. et al (2016) Biophysical Journal
9. A cryogenic, coincident fluorescence, electron and ion beam microscope Boltje, D. et al (2022) eLife