Cryogenic Focused Ion Beam-Scanning Electron Microscopy (Cryo-FIB-SEM) allows scientists to peer deep into the internal architecture of materials and biological specimens at nanoscale resolution. This technique captures three-dimensional structure while preserving the sample in a state as close as possible to its natural condition. By combining a focused ion beam and a scanning electron microscope within a cryogenic environment, researchers obtain volumetric data for complex systems.
The Need for Cryo-Preparation
Visualizing delicate samples, especially biological cells and soft materials, under the high-vacuum conditions of an electron microscope presents a significant challenge. Traditional preparation methods, such as chemical fixation and dehydration, introduce structural artifacts that distort the sample’s true appearance. Chemical fixatives alter the shape and position of cellular components, while drying causes severe shrinkage and collapse of fragile structures like membranes and proteins.
To circumvent these issues, Cryo-FIB-SEM relies on cryogenic preparation, which involves ultrarapid freezing of the sample. This flash-freezing process, often achieved by plunging the sample into liquid ethane cooled by liquid nitrogen, must occur fast enough to prevent the formation of destructive ice crystals. When water freezes slowly, it forms hexagonal crystalline ice, which has sharp edges that pierce and destroy cellular structures.
Rapid freezing, known as vitrification, fixes the water in a non-crystalline, glass-like solid state, known as vitreous ice. This vitreous state is attained when the cooling rate exceeds a million degrees Celsius per second, preserving the native cellular ultrastructure with minimal distortion. Maintaining the sample below -130°C throughout the imaging process ensures this near-native state is preserved.
Creating the View: Focused Ion Beam Milling
The “FIB” component, a Focused Ion Beam, enables the high-precision sculpting of the frozen sample. The FIB acts like a nanoscale sandblaster, using a tightly focused beam of heavy ions—typically Gallium—to precisely ablate, or mill, away material. In a Cryo-FIB-SEM instrument, the ion column and the electron column (SEM) are positioned to intersect their beams at the sample surface, allowing for simultaneous cutting and imaging.
The primary role of the FIB is to create a lamella, which is an ultrathin, electron-transparent slice of the frozen sample. For a subsequent technique like cryo-electron tomography, this lamella must be thinned down to a thickness between 100 and 300 nanometers, which is thin enough for electrons to pass through. The milling process is performed in several steps, starting with a rough cut to remove bulk material and ending with a fine polishing step to achieve the final nanometer-scale thickness.
The FIB can also be used in a sequential “slice-and-view” mode to acquire a three-dimensional volume of a thicker sample. In this technique, the ion beam precisely removes an extremely thin layer of material, often between 3 and 10 nanometers thick, exposing a fresh surface. The scanning electron microscope then images this newly exposed face, and the process is repeated dozens or hundreds of times. This cyclical removal and imaging generates a stack of high-resolution images, which are then computationally aligned and processed to reconstruct the full 3D volume of the sample.
Visualizing Structure: Scanning Electron Microscopy
The “SEM” component, the Scanning Electron Microscope, is responsible for acquiring the high-resolution image data after the FIB has prepared the surface. Unlike traditional light microscopes that use visible light, the SEM uses a finely focused beam of electrons to scan the sample’s surface. As the electron beam interacts with the atoms in the sample, it generates various signals, most commonly secondary and backscattered electrons.
In the slice-and-view technique, the electron beam scans the freshly milled face of the frozen sample, and detectors capture the emitted electrons. Contrast in the resulting image is generated by differences in the sample’s density and elemental composition. Because the sample is frozen and unstained, contrast comes from the inherent differences between cellular components, such as membranes and cytoplasm, allowing visualization of the cell’s natural organization.
Each image captured by the SEM represents a thin cross-section of the sample volume. By repeating the FIB milling and SEM imaging cycle, a complete stack of images is acquired, with each image precisely registered to the previous one. Researchers then use specialized software to digitally align and render these two-dimensional slices into a three-dimensional model, enabling volumetric analysis of the sample’s internal structure.
Breakthrough Applications in Science
Cryo-FIB-SEM has driven advancements across life sciences and materials research by enabling the visualization of structures in their near-native state. In cell biology, the technique is routinely used for cellular tomography, providing high-resolution, three-dimensional maps of entire cells and their organelles. Researchers can visualize the intricate network of the endoplasmic reticulum or the organization of mitochondria within a human cell with nanometer precision.
The technology is also instrumental in studying the interaction between pathogens and host cells in situ, revealing how viruses and bacteria invade and manipulate cellular machinery. This allows scientists to see the exact moment a virus, like SARS-CoV-2, forms its replication compartments inside a host cell, providing structural context for drug development. In materials science, Cryo-FIB-SEM is applied to analyze sensitive materials like lithium-ion battery components and polymers, which are easily damaged or deformed by conventional room-temperature preparation.
The technique allows for the generation of lamellae from thick tissues or large cells. These lamellae can then be transferred to a cryo-transmission electron microscope for higher-resolution imaging of individual macromolecules. This workflow links the architecture of an entire cell to the structure of its constituent proteins, providing a comprehensive view of biological systems.