Eukaryotic cells have numerous cellular structures, including a variety of organelles and macromolecular complexes. These structures have specific physiological functions and work interactively to perform certain cellular activities. Therefore, studying these structures in their native state is essential to understand the real physiological processes in the cells. In situ investigation of cellular structures does not only provide morphology, distribution, and abundance information, but also reveals their interaction mechanisms, thereby providing new insights into the understanding of life.

Cryo-electron tomography (cryo-ET) is currently the principal technique to resolve the in situ structures of biological specimens. By collecting tilted series of transmission electron images and performing image reconstruction, cryo-ET determines the 3D structures of bio-specimens with a nanometer-level resolution. A prerequisite for applying cryo-ET is to fix the sample under cryogenic conditions. High-pressure freezing and plunge freezing are well-established cryo-fixation methods that preserve biological specimens in their near-native state in vitreous ice. Benefiting from these techniques, cryo-ET has been widely applied to cells and tissues.

One limitation to cryo-ET is its restricted imaging depth, which is typically a few hundred nanometers owing to the confined penetration capabilities of electrons. Therefore, reducing the thickness of the samples to that of lamellae of approximately 200 nm is necessary before applying cryo-ET. Focused ion beam (FIB) milling has been recently employed to prepare lamellae of bio-specimens for cryo-ET. Compared to traditional ultramicrotomy, FIB milling avoids artifacts such as distortions, crevasses, and compression when fabricating the lamella. However, conventional FIB milling does not allow site-specific milling, because in a dual-beam FIB/SEM system, FIB or SEM image only illustrates the surface morphology of the sample and cannot provide more information to recognize and localize the underlying interest targets. When milling cells with FIB, cutting at an arbitrary position can only hit abundant cellular structures such as Golgi apparatus or mitochondria but cannot be used to prepare lamellae containing specific targets. This drawback hinders the application of FIB in cryo-ET.

The “blind” milling can be improved by correlative light and electron microscopy (CLEM). In CLEM, the targets of interest are fluorescently labelled and can be identified by fluorescence imaging. After registering the light and FIB images, fluorescence signal can be used to guide the FIB to mill at specific sites. Currently, various light imaging modalities have been adopted to navigate FIB fabrication, including widefield microscopy, confocal microscopy, and Airyscan. Moreover, two major working routines, that is, pipelined and integrated workflows, have been established to perform fluorescence-guided FIB milling. Therefore, it is important and necessary to summarize the existing techniques and discuss the advantages and limitations of different working routines to provide guidelines for researchers to choose the appropriate protocols.

This study reviews the essential techniques involved in fluorescence-guided cryo-FIB milling. First, plunge freezing is introduced. Plunge freezing is the most commonly used technique to vitrify cells. The key aspects to obtain good plunge-frozen specimens are discussed, including the choice of electron microscope grids and supporting films (Fig. 2), available commercial instruments (Fig. 3), and standard protocols.

Second, as a popular method to prepare lamellae of vitrified cells, FIB milling is discussed in several aspects: the working principle is introduced; the relevant instrumentations are summarized, including dual-beam FIB/SEM system (Fig. 4), cryostage and cryotransfer systems (Fig. 5), and Autogrid and sample holder (Fig. 6); and the milling of frozen cells is outlined (Fig. 7).

Third, the principle (Fig. 8) and workflow (Fig. 9) of fluorescence-guided FIB milling is introduced. Pipelined and integrated workflows are described, and relevant commercial instruments are overviewed (Figs. 10 and 11). The different workflows and various systems are compared (Table. 1). The most recent developments of integrated solutions are discussed in detail. Sun Fei's research group and Ji Wei's research group from the Institute of Biophysics, Chinese Academy of Sciences have developed novel integrated light, ion, and electron microscopies (Figs. 12 and 13), thereby providing new avenues for performing accurate and efficient FIB milling at specific sites under fluorescence guidance.

In situ investigation of cellular structures using cryo-ET has recently become an interesting research topic. Fluorescence-guided FIB milling has been applied to mill vitrified biological samples at specific sites. The recent developments in integrated cryo-FLM-FIB/SEM systems and workflows provide efficient and accurate methods to fabricate cell lamellae containing desired targets. These innovations have the potential to serve as all-in-one solutions for site-specific cryo-lamella preparation for cryo-ET in the future.