Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells

Cryogenic focused ion beam milling with scanning electron microscopy (CryoFIB-SEM) has become the dominant approach for preparing sub-200-nm thick lamellae of cells for cryogenic electron tomography (CryoET). Recently, fluorescence microscopy has been incorporated into CryoFIB-SEM systems to guide lamella production toward fluorescently labeled targets. However, in most implementations, the optical and ion beam imaging systems have distinct focal planes in different regions of the vacuum chamber-functioning effectively as two separate microscopes. This configuration requires image registration between modalities, which often lacks the accuracy needed to reliably target small, rare structures in situ. We present a workflow based on a customized tri-coincident imaging platform that enables simultaneous fluorescence imaging and focused ion beam milling. This allows for real-time fluorescence feedback that can be used to determine when to terminate milling in order to preserve fluorescently labeled structures of interest in the final lamella. Using this approach, we targeted the microtubule-organizing center (MTOC) in vitrified macrophages and achieved a >60% success rate, confirmed by subsequent cryogenic electron tomography and correlative light and electron microscopy.