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bioRxiv · 10.64898/2026.10.28.761815

Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography

Smith, J. L. R.·Gannon, R. N.·Glynn, C.·Naismith, J. H.·Filevich, J.·Grange, M.
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Abstract

In situ electron cryo-tomography (cryo-ET) is a powerful tool for visualising biology inside cells at the molecular scale. The routine application of cryo-ET in tissue samples promises biological insight beyond single cells, but is currently limited by the technical challenge of preparing high quality, electron-transparent specimens from thick (>100 m) biopsies efficiently. Here, we demonstrate that cryogenic femtosecond pulsed laser ablation (cryo-fs-PLA) can accelerate the preparation of vitreous specimens of mammalian brain tissue for cryo-ET. Using cryogenic fluorescence microscopy, regions of interest within high-pressure frozen tissue can be specifically targeted, and cryo-fs-PLA can be employed as a preparative step for serial cryogenic lift-out experiments. We define cryo-fs-PLA parameters and determine ablation rates for two different laser wavelengths, and demonstrate that tilt series collected on lamellae fabricated from sites exposed to cryo-fs-PLA largely maintain their vitrification state. Incorporating cryo-fs-PLA into existing cryo-ET workflows has the potential to unlock the sample preparation of tissues at the throughput necessary for routine in situ cell biology of multicellular samples.

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Smith, J. L. R., Gannon, R. N., Glynn, C., Naismith, J. H., Filevich, J., Grange, M.. 2026-10-28. Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography. https://doi.org/10.64898/2026.10.28.761815

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