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Square condenser apertures for square cameras in low-dose transmission electron microscopy

Nature Methods volume 21, pages 566–568 (2024)Cite this article

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Abstract

In transmission electron microscopy (TEM), cameras are square or rectangular but beams are round so the circular lobes irradiate adjacent areas, precluding further neighboring acquisition for beam-sensitive samples. We present condenser aperture plates with square and rectangular shapes that improve the efficiency of area usage by 70% and enhance montage imaging for beam-sensitive specimens. We demonstrate the compatibility of these condenser aperture plates with high-resolution cryogenic TEM by reconstructing a 1.8-Å map of equine apo-ferritin.

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Fig. 1: Single-particle cryo-TEM images are acquired in an ice-covered foil hole.
Fig. 2: Biological macromolecules reconstructed from single-particle cryo-EM data recorded with square and rectangular condensor apertures.

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Data availability

We have a limited number (about 50) of spare aperture plates from the initial manufacturing run that we can give to people interested in incorporating them into their microscopes. All cryo-EM data are available upon request and refined maps have been uploaded to the EMDB with the accession codes EMD-41933, EMD-41938, EMD-41936, EMD-41937 and EMD-42882 for the Gatan K3 round beam and rectangular beam apoferritin map, the Falcon IV round beam and square beam apoferritin map, and the AmyR1:sCT map, respectively.

Code availability

All in-house software is available in the supplementary materials and in the GitHub repositories https://github.com/HamishGBrown/ApertureplateDesigner (for the aperture generation script) and https://github.com/HamishGBrown/EPU_group_AFIS (for the script to import optics groups to Cryosparc).

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Acknowledgements

We thank P. Francis, a staff member of the Ian Holmes Imaging Centre, for training and assistance with the ThermoFisher Teneo VolumeScope SEM. We thank S. Zeltmann, J. Ciston and C. Ophus from the National Centre for Electron Microscopy (NCEM) at Lawrence Berkeley National Laboratory for initial discussions on fabrication of condenser aperture plates for ThermoFisher Titan TEMs and the several apertures in a single plate design was inspired by their example. We thank M. Belousoff for providing vitrified grids of AMYR1-sCT. ThermoFisher Krios G4 and Artica G2 TEMs and a ThermoFisher VolumeScope SEM located at the Ian Holmes Imaging Centre in the Bio21 MolecularScience and Biotechnology Institute of the University of Melbourne were used in this paper. This work was performed in part at the Melbourne Centre for Nanofabrication in the Victorian Node of the Australian National Fabrication Facility. The next generation of aperture plates are fabricated by Norcada (https://www.norcada.com/), a micro-electro-mechanical systems and photonics product company; we also thank Norcada for initial discussions on aperture fabrication in the initial stages of the project. In the late stages of manuscript preparation, we became aware of the work of Chua et al.18, which is similar to the work presented here. H.G.B. acknowledges funds from a University of Melbourne Early Career Researcher Award.

Author information

Authors and Affiliations

  1. Ian Holmes Imaging Centre and ARC Industrial Training Centre for Cryo Electron Microscopy of Membrane Proteins, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia

    Hamish G. Brown & Eric Hanssen

  2. Melbourne Centre for Nanofabrication, Australian National Fabrication Facility, Clayton, Victoria, Australia

    Dan Smith

  3. Thermo Fisher Scientific Australia, Scoresby, Victoria, Australia

    Benjamin C. Wardle

  4. Department of Biochemistry and Pharmacology, The University of Melbourne, Parkville, Victoria, Australia

    Eric Hanssen

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  1. Hamish G. Brown
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Contributions

H.G.B. and E.H. conceived the study, recorded the cryo-EM data with round and square beams, performed data analysis and wrote the manuscript. H.G.B. designed the apertures and aligned them once installed in the microscope. D.S. fabricated H.G.B.’s designs. B.C.W. advised on aperture plate specifications for ThermoFisher TEMs and installed the aperture plates into the microscope.

Corresponding author

Correspondence to Hamish G. Brown.

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Competing interests

B.C.W. is a senior field service engineer of ThermoFisher Scientific, the company that manufactures the Talos Arctica and Titan Krios Cryo-TEMs. The other authors declare no competing interests.

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Peer review information

Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Discussion and Tables 1 and 2.

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Supplementary Software 1

Contains local copies of GitHub repositories https://github.com/HamishGBrown/AperturePlateDesigner and get_exp_id_from_star.py from https://github.com/HamishGBrown/EPU_group_AFIS for reader convenience.

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Brown, H.G., Smith, D., Wardle, B.C. et al. Square condenser apertures for square cameras in low-dose transmission electron microscopy. Nat Methods 21, 566–568 (2024). https://doi.org/10.1038/s41592-024-02206-9

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