High Accuracy Fluorescence Guided Focused Ion Beam Milling

Cryo-electron tomography (cryo-ET) is a powerful approach for visualizing macromolecular structures directly within cells, but its broader application is limited by the difficulty of reliably targeting specific structures for imaging. In particular, capturing small or rare objects within FIB-milled lamellae remains a major bottleneck. Here, we establish fluorescence-guided cryo-FIB milling workflows that overcome key sources of targeting error and enable routine capture of structures across a wide size range. For larger objects (>500 nm), we develop a single step registration-based targeting strategy that combines FIB-milled fiducials with physically grounded depth correction to account for focal shifts arising from refractive index mismatch. For smaller targets (150-500 nm), we implement real-time fluorescence-guided milling on a commercially available FIB SEM instrument with an integrated cryo fluorescence microscope allowing dynamic monitoring and precise termination of milling at the onset of target ablation. Using this strategy, we achieve consistent recovery of lamellae containing the targeted structure, including small single-copy organelles such as centrioles and cilia. Together, these workflows expand the accessible target space for cryo-ET and provide practical solutions for studying cellular structures that have previously been difficult to capture.