Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography
- 1Rosalind Franklin Institute
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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BibTeX
@misc{smith2026femtosecond,
title = {{Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography}},
author = {J. L. R. Smith and R. N. Gannon and C. Glynn and J. H. Naismith and J. Filevich and M. Grange},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.28.761815},
url = {https://www.biorxiv.org/content/10.64898/2026.10.28.761815v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography
AU - J. L. R. Smith
AU - R. N. Gannon
AU - C. Glynn
AU - J. H. Naismith
AU - J. Filevich
AU - M. Grange
PY - 2026
DA - 2026/10/28
PB - bioRxiv
DO - 10.64898/2026.10.28.761815
UR - https://www.biorxiv.org/content/10.64898/2026.10.28.761815v1
AB - 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.
ER -