Cryo-focused ion beam (cryo-FIB) milling is indispensable for preparing thin lamellae from thick biological samples for cryo-electron tomography (cryo-ET), especially at the tissue level. However, existing approaches remain technically demanding and susceptible to lamella instability. We present a detailed, optimized serial lift out cryo-FIB milling protocol specifically designed for multicellular tissue samples, emphasizing improved lamella stability, reproducibility, and reduced operator dependency. This method integrates six-sided attachment geometry and adjustable lamella slit spacing, enabling efficient lift-out and precise reattachment of variable-sized tissue volumes to pre-milled grids. The procedure is applicable across a range of biological tissues and is compatible with integrated cryo-correlative light and electron microscopy (cryo-CLEM) pipelines. By enhancing structural preservation and simplifying operational steps, this protocol provides a robust, reproducible framework to facilitate high-throughput, in situ structural analysis of tissues by cryo-ET.
Optimized Serial Lift-Out Cryo-FIB Technique Unveiled
Higher Education Press
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