Workflow and instrumentation for multiscale correlative imaging in voluminous cryogenic specimens

Cryo-electron tomography (cryo-ET) enables molecular-resolution imaging of intracellular environments, but is constrained by the need to generate electron-transparent sections of the biological specimen and thus a limited imaging volume. To alleviate such limitations, we present a multiscale correlative workflow that links cryo-confocal fluorescence microscopy (cryo-FLM), cryo-focused ion beam-scanning electron microscopy (cryo-FIB-SEM) volume imaging (cryo-vEM), and cryo-FIB serial lamellae lift-out for cryo-ET in high-pressure frozen voluminous specimens. An integral part of this workflow is a sample holder that preserves specimen orientation across instruments, reduces manual handling, and overcomes stage-tilt limitations in cryo-FIB-SEM hardware. Applied to vitrified colorectal cancer organoids, we demonstrate that this workflow enables fluorescence-guided site preparation for cryo-vEM, which provides biological context beyond the limited field of view of cryo-ET, and targeted lift-out for fabrication of multiple lamellae from defined regions of interest. The reconstructed tomograms reveal subcellular architecture and cell-cell interfaces. This workflow establishes a modular methodological framework for continuum-resolution cryogenic imaging of complex 3D biological specimens.