Radovan Spurny, Thermo Fisher Scientific, Eindhoven, Netherlands

Plasma focused ion beam (PFIB) milling has become a key enabling technology for in situ structural biology, allowing researchers to prepare lamellae from vitrified cells and tissues for cryo-electron tomography (cryo-ET). As cryo-ET moves towards wider adoption and increasingly complex biological questions, there is growing demand for workflows that combine precise targeting, reproducible lamellae preparation, and higher throughput.

In this workshop, we will explore recent advances in PFIB-based cryo-lamella preparation and their impact on structural biology workflows. We will cover correlative approaches that bridge fluorescence microscopy and cryo-ET, enabling researchers to identify and target specific cellular structures prior to milling, including how integrated fluorescence microscopy (iFLM) can be used to localize fluorescently labelled features within vitrified specimens and guide the preparation of lamellae enriched for biological targets of interest.

Through workflow demonstrations, participants will follow the journey from frozen sample to tomography-ready lamella, including sample navigation, correlative targeting, automated milling strategies, and transfer to the transmission electron microscope. We will also discuss recent developments in user-guided automation and multi-grid processing that are helping to improve reproducibility while reducing the hands-on time required for cryo-FIB preparation.The workshop will cover practical considerations for implementing cryo-PFIB workflows, and provide an opportunity to discuss how advances in correlative imaging and automation are helping bridge the gap between cellular context and molecular structure.