BPS2026 – Fiducial-free guidance for cryo-FIB lamellae production enabled by simultaneous fluorescence microscopy and ion milling

Cryogenic focused ion beam (cryo-FIB) milling has become a ubiquitous step in cryogenic electron tomography (cryo-ET) for imaging cells and tissues. Two challenges facing this approach that limit throughput and biological insight are (1) ensuring that the final electron-semi-transparent lamella actually contains the rare, nanoscale structures of interest and (2) precisely localizing target proteins within that lamella to guide tilt-series acquisition and enable protein identification in final reconstructions. I will present our group’s solutions based on integrating advanced optical microscopy directly into the FIB-SEM in a coincident geometry. Coincident optical and ion imaging enables true simultaneous light microscopy during milling. This configuration revealed an interferometric optical signal arising from reflection of the excitation light off the evolving lamella surface; exploiting this signal allows us to guide milling with tens-of-nanometer accuracy without fiducials or registration. I will also describe our ongoing integration of super-resolution fluorescence modalities into the FIB-SEM to achieve accurate protein localization in the final lamellae. Taken together, these efforts make significant progress on open challenges in the FIB milling workflow.