Nanomachines and ultrastructures inside cells are the basic units involved in life activities. They perform specific physiological functions through close cooperation with each other. Seeing is believing. Studying the in situ assembly and function of these complicated and precise nanostructures has been a hot topic in life science. Cryo-electron tomography (cryo-ET) is currently the main technique for in situ structural analysis.

Due to the limitation of electron beam penetration, cell and tissue samples should be milled to lamella of ~200 nanometers using focused ion beams (FIB) for imaging. However, the random milling technique brings great challenges to the study of specific targets with relatively low abundance in cells. The prepared cryo-lamella sample often fails to retain the target of interest.

To deal with this bottleneck, teams from Institute of Biophysics of the Chinese Academy of Sciences developed two different types of precise systems to prepare target cryo-lamellae. They proposed a scheme to use fluorescence imaging to navigate cryo-FIB milling, and separately developed the integrated correlative light and electron microscopy (CLEM) cryo-FIB system, named as ELI-TriScope and CLIEM respectively. The studies were both published in Nature Methods.

The ELI-TriScope system uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embeds an optical imaging system (cryogenic SimulTAneous monitoR system, cryo-STAR) just underneath the vitrified specimen. It sets electron, light, and ion beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. Cryo-FIB milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule.

The CLIEM system incorporates a 3D multi-color confocal microscope into a dual-beam FIB-SEM system. Through high-quality whole-cell multicolor 3D fluorescence imaging, the 3D spatial position can be accurately located. Through ingenious projection transformation, the light and FIB images can be rapidly and accurately correlated, achieving FIB milling with a high precision of tens of nanometers. Also, a "virtual lamella" function that generates virtual lamella of specific positions in the cell is developed, which helps to determine the optimal milling site in a crowded cell.

Using the above systems, the teams efficiently prepared cryo-lamellae of cells. Through subsequent cryo-electron tomography (cryo-ET) analysis, they discovered new in situ structural features of human centrosomes, and studied cellular ultrastructures such as organelle interaction sites.

These results demonstrated that ELI-TriScope and CLIEM, as efficient and precise cryo-FIB techniques, provide new solutions for site-specific sample preparation of cryo-ET, which have wide potential applications in the study of the ultrastructure of specific events in cells and are expected to advance the development of in situ structural biology.