Rapid femtosecond laser trenching accelerates cryo-electron tomography lamella workflows
- 1Diamond Light Source
Abstract
Transmission electron microscopy requires electron-transparent specimens, typically below 200 nm thick. For cellular and tissue samples this is typically achieved by focused ion-beam (FIB) thinning to form lamellae. Milling times scale with specimen thickness, and high-pressure-frozen specimens more than 10 m thick can take up to 2 hours per lamella. We present a pre-processing approach in which bulk material is removed by ultrashort-pulse laser ablation prior to milling, rapidly generating large trenches in vitrified grids under cryogenic conditions and leaving material to be finished by conventional milling to yield electron-transparent lamellae. Laser ablation completes in seconds (4 seconds per pattern), compared with minutes to tens of minutes for FIB trench milling, and introduces negligible damage to the final lamellae. By replacing the most time-intensive, contamination-sensitive bulk-removal steps with seconds-long laser trenching, this open-source approach reduces time-to-lamellae, increasing throughput and preserves compatibility with standard cryo-FIB/SEM polishing and cryo-ET data-collection pipelines.
Related
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- Whole cryo-FIB lamella acquisition at high magnification with rectangular beam montage cryo-electron tomography
BibTeX
@misc{orr2026rapid,
title = {{Rapid femtosecond laser trenching accelerates cryo-electron tomography lamella workflows}},
author = {C. M. Orr and P. N. M. Shah and E. C. Bragginton and S. Chakraborty and H. M. E. Duyvesteyn and S. Riggs and D. I. Stuart and A. Wagner},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.23.761368},
url = {https://www.biorxiv.org/content/10.64898/2026.10.23.761368v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Rapid femtosecond laser trenching accelerates cryo-electron tomography lamella workflows
AU - C. M. Orr
AU - P. N. M. Shah
AU - E. C. Bragginton
AU - S. Chakraborty
AU - H. M. E. Duyvesteyn
AU - S. Riggs
AU - D. I. Stuart
AU - A. Wagner
PY - 2026
DA - 2026/10/23
PB - bioRxiv
DO - 10.64898/2026.10.23.761368
UR - https://www.biorxiv.org/content/10.64898/2026.10.23.761368v1
AB - Transmission electron microscopy requires electron-transparent specimens, typically below 200 nm thick. For cellular and tissue samples this is typically achieved by focused ion-beam (FIB) thinning to form lamellae. Milling times scale with specimen thickness, and high-pressure-frozen specimens more than 10 m thick can take up to 2 hours per lamella. We present a pre-processing approach in which bulk material is removed by ultrashort-pulse laser ablation prior to milling, rapidly generating large trenches in vitrified grids under cryogenic conditions and leaving material to be finished by conventional milling to yield electron-transparent lamellae. Laser ablation completes in seconds (4 seconds per pattern), compared with minutes to tens of minutes for FIB trench milling, and introduces negligible damage to the final lamellae. By replacing the most time-intensive, contamination-sensitive bulk-removal steps with seconds-long laser trenching, this open-source approach reduces time-to-lamellae, increasing throughput and preserves compatibility with standard cryo-FIB/SEM polishing and cryo-ET data-collection pipelines.
ER -