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

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.