Cryo-focused ion beam (cryo-FIB) milling has become a key method for preparing lamellae that enable visualization of native protein structures and cellular ultrastructure by cryo-electron tomography (cryo-ET). A major step toward studying specific proteins or subcellular regions is fluorescence-guided targeting during milling. Yet, establishing precise registration between fluorescence microscopy (FM) and cryo-FIB remains a persistent challenge. In this work, we introduce two complementary approaches to improve fluorescence guidance in cryo-FIB workflows. First, we systematically assessed registration accuracy in FIB-SEM systems where FM and ion/electron beams are positioned at different geometries, requiring stage shifts to alternate between modalities. By identifying and quantifying the dominant sources of registration error, we developed correction protocols that increase the precision of aligning FM-identified regions with subsequent milling sites. While prior studies demonstrated that simultaneous FM imaging during milling can enhance targeting accuracy by avoiding error-prone registration steps, such capability is not yet commercially available. The geometry of the Thermo Fisher Scientific Arctis FIB-SEM offers coincidence between the light microscope and the ion beam, but it does not natively support simultaneous imaging. We modified operating procedures to incorporate real-time FM feedback, enabling iterative refinement of target selection during milling through open-source software tools. Using this strategy, we achieved markedly improved targeting of primary cilia—a small and infrequent organelle—for downstream cryo-ET studies. Together, these methods establish a more robust framework for site-specific lamella preparation across a range of cryo-FIB-SEM platforms, thereby enhancing the efficiency of cryo-ET workflows for investigating rare and small biological structures.
Peukes et al. (Sun,) studied this question.