Reduction of SEM charging artefacts in native cryogenic biological samples

Abner Velazco, Thomas Glen, Sven Klumpe, Avery Pennington, Jianguo Zhang, Jake L. R. Smith, Calina Glynn, William Bowles, Maryna Kobylynska, Roland A. Fleck, James H. Naismith, Judy S. Kim, Michele C. Darrow, Michael Grange, Angus I. Kirkland and Maud Dumoux ()
Additional contact information
Abner Velazco: Harwell Science & Innovation Campus
Thomas Glen: Harwell Science & Innovation Campus
Sven Klumpe: Max Planck Institute of Biochemistry
Avery Pennington: Harwell Science & Innovation Campus
Jianguo Zhang: Harwell Science & Innovation Campus
Jake L. R. Smith: Harwell Science & Innovation Campus
Calina Glynn: Harwell Science & Innovation Campus
William Bowles: Harwell Science & Innovation Campus
Maryna Kobylynska: King’s College London
Roland A. Fleck: King’s College London
James H. Naismith: Physical and Life Science Division
Judy S. Kim: Harwell Science & Innovation Campus
Michele C. Darrow: Harwell Science & Innovation Campus
Michael Grange: Harwell Science & Innovation Campus
Angus I. Kirkland: Harwell Science & Innovation Campus
Maud Dumoux: Harwell Science & Innovation Campus

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging of subcellular structures at the mesoscale in a representation of their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional model of cells and tissues. The correlation of specific regions of interest with cryo-electron microscopy (cryoEM) can additionally enable subsequent high-resolution analysis. However, the use of serial FIB/SEM imaging-based methods is often limited due to charging artefacts arising from insulating areas of cryogenically preserved samples. Here, we demonstrate the use of interleaved scanning to attenuate these artefacts, allowing the observation of biological features that otherwise would be masked or distorted. We apply our method to samples where inherent features were not visible using conventional scanning. These examples include membrane contact sites within mammalian cells, visualisation of the degradation compartment in the algae E. gracilis and observation of a network of membranes within different types of axons in an adult mouse cortex. The proposed alternative scanning method could also be applied to imaging other non-conductive specimens in SEM.

Date: 2025
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DOI: 10.1038/s41467-025-60545-3

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