CryoCLEM

CryoCLEM, or cryo-correlative light and electron microscopy, is a workflow that combines cryogenic fluorescence light microscopy (cryo-FLM) with cryogenic electron microscopy (cryo-EM) to study biological specimens preserved in a vitrified, near-native state. The aim is to locate fluorescently labeled structures within the high-resolution ultrastructure provided by electron microscopy, enabling direct correlation between molecular identity and cellular architecture.

The general workflow involves preparing specimens by rapid vitrification, such as plunge freezing or high-pressure freezing, onto electron microscopy grids. Fluorophores or fluorescent proteins label target components, and cryo-fluorescence imaging identifies regions of interest at liquid-nitrogen temperatures. Correlation then aligns the fluorescence data with electron microscopy maps using fiducials visible in both modalities (for example, fluorescent or electron-dense beads) or distinctive landmarks, often with specialized software to achieve precise alignment.

Imaging proceeds with cryo-EM on the correlated regions. If needed, sample thinning by cryo-focused ion beam milling creates electron-transparent lamellae suitable for cryo-tomography (cryo-ET), providing three-dimensional ultrastructural context. The resulting data allow localization of fluorescent signals within 3D organellar or molecular structures and can support sub-tomogram averaging to resolve complexes.

Applications span virology, organelle and cytoskeleton studies, endocytosis, and protein complex investigations within intact cellular environments. Advantages include preserving native molecular organization and linking fluorescence-based identity to high-resolution structure. Challenges involve maintaining fluorescence under cryogenic conditions, minimizing radiation damage, achieving accurate alignment, and managing the technical complexity of integrated instrumentation and workflows.