Rapid femtosecond laser trenching accelerates cryo-electron tomography lamella workflows
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…
Transmission electron microscopy demands electron-transparent specimens, generally thinner than 200 nanometers. To achieve this for cellular and tissue samples, focused ion-beam (FIB) thinning is often employed to produce lamellae. However, milling times increase with specimen thickness, and frozen samples over 10 meters can require up to two hours per lamella.
In this study, researchers introduce a pre-processing technique that utilizes ultrashort-pulse laser ablation to remove bulk material before milling, forming large trenches in frozen grids at cryogenic temperatures. This method generates the lamellae in mere seconds (4 seconds per pattern) as opposed to minutes to tens of minutes for FIB trench milling.
Importantly, laser ablation causes minimal damage to the final lamellae. By substituting the most time-consuming and contamination-sensitive bulk-removal steps with laser trenching, this open-source approach enhances time-to-lamellae, thereby increasing throughput. Furthermore, the technique remains compatible with standard cryo-FIB/SEM polishing and cryo-ET data-collection protocols.
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