Depth-resolved analysis reveals target-dependent cryo-FIB damage profiles

Abstract

Cryogenic focused ion beam (cryo-FIB) milling enables cellular cryo-electron tomography but can compromise high-resolution structural information near milled surfaces. Here we establish recombinant human apoferritin (apoF) as a reporter of depth-dependent structural preservation in cellular lamellae. ApoF yielded a 2.05 [A] in situ reconstruction. Apparent FIB damage was most pronounced within the first 15 nm for xenon, 30 nm for gallium, 45 nm for argon and remained detectable at least 75 nm from oxygen-milled surfaces under the workflows tested. Endogenous 70S ribosomes in the same xenon-milled tomograms showed a broader 30-45 nm profile, whereas {beta}-galactosidase showed a similar narrow xenon-associated profile in a different specimen. Xenon-milled lamellae 125-150 nm thick yielded the highest resolution among the thickness groups examined, with lower resolution in the thinner and thickest groups. These findings show structural preservation is constrained by both surface-associated damage and lamella thickness, with the apparent extent of damage varying by molecular reporter. Previous ribosome-based surface-exclusion estimates may therefore be overly conservative for some molecular targets.

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BibTeX RIS

BibTeX

@misc{nwokonko2026depthresolved,
  title = {{Depth-resolved analysis reveals target-dependent cryo-FIB damage profiles}},
  author = {R. M. Nwokonko and M. Z. Qayyum and D. Karia and V. Maingi and A. Rai and Y. Sheng and S. A. Haque and C. Thompson and S. Sviben and H. Yanagisawa and J. M. de la Rosa-Trevin and R. C. Kalathur and G. C. Skiniotis and A. Kotecha},
  howpublished = {bioRxiv},
  year = {2026},
  month = sep,
  doi = {10.64898/2026.09.16.752186},
  url = {https://www.biorxiv.org/content/10.64898/2026.09.16.752186v1},
  note = {CC BY},
}

RIS

TY  - UNPB
TI  - Depth-resolved analysis reveals target-dependent cryo-FIB damage profiles
AU  - R. M. Nwokonko
AU  - M. Z. Qayyum
AU  - D. Karia
AU  - V. Maingi
AU  - A. Rai
AU  - Y. Sheng
AU  - S. A. Haque
AU  - C. Thompson
AU  - S. Sviben
AU  - H. Yanagisawa
AU  - J. M. de la Rosa-Trevin
AU  - R. C. Kalathur
AU  - G. C. Skiniotis
AU  - A. Kotecha
PY  - 2026
DA  - 2026/09/16
PB  - bioRxiv
DO  - 10.64898/2026.09.16.752186
UR  - https://www.biorxiv.org/content/10.64898/2026.09.16.752186v1
AB  - Cryogenic focused ion beam (cryo-FIB) milling enables cellular cryo-electron tomography but can compromise high-resolution structural information near milled surfaces. Here we establish recombinant human apoferritin (apoF) as a reporter of depth-dependent structural preservation in cellular lamellae. ApoF yielded a 2.05 [A] in situ reconstruction. Apparent FIB damage was most pronounced within the first 15 nm for xenon, 30 nm for gallium, 45 nm for argon and remained detectable at least 75 nm from oxygen-milled surfaces under the workflows tested. Endogenous 70S ribosomes in the same xenon-milled tomograms showed a broader 30-45 nm profile, whereas {beta}-galactosidase showed a similar narrow xenon-associated profile in a different specimen. Xenon-milled lamellae 125-150 nm thick yielded the highest resolution among the thickness groups examined, with lower resolution in the thinner and thickest groups. These findings show structural preservation is constrained by both surface-associated damage and lamella thickness, with the apparent extent of damage varying by molecular reporter. Previous ribosome-based surface-exclusion estimates may therefore be overly conservative for some molecular targets.
ER  -

Abstract

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