Cryo-CLEM, or Cryogenic Correlative Light and Electron Microscopy, is a specialized imaging technique that bridges the gap between different levels of biological observation. It allows scientists to gain a comprehensive understanding of biological structures and processes by combining two powerful microscopy methods. The overarching purpose of Cryo-CLEM is to provide both broad contextual information about a cell or tissue and highly detailed, ultra-high-resolution views of specific components within it. This integrated approach helps researchers visualize complex biological events and molecular arrangements in a way that neither technique could achieve on its own.
The Need for Correlative Microscopy
Traditional microscopy methods often provide only a partial view of biological samples, necessitating a more integrated approach for complete understanding. Light microscopy (LM) excels at observing living cells, offering a large field of view and the ability to visualize specific molecules tagged with fluorescent markers. This provides excellent contextual information about cellular processes and the location of various components. However, LM has a resolution limit of approximately 200 nanometers, meaning it cannot resolve fine structures like individual proteins or cellular membranes with sufficient detail.
Electron microscopy (EM), in contrast, provides images with extremely high resolution, revealing intricate ultrastructural details of cells, sometimes down to the sub-nanometer scale. This allows for the visualization of organelles and macromolecular complexes with remarkable clarity. A significant limitation of EM, however, is that samples typically require extensive preparation, including chemical fixation and dehydration, which can alter their native state and usually kills the cell, making live imaging impossible. Furthermore, identifying specific molecules within the dense EM image without prior labeling can be challenging.
These individual strengths and weaknesses created a gap in biological research. Scientists needed to correlate the dynamic, contextual information from LM with the high-resolution structural details from EM on the exact same sample and location. Cryo-CLEM was developed to address this need, combining functional insights from fluorescently labeled structures with their precise ultrastructural context.
The Cryo-CLEM Process
The Cryo-CLEM workflow involves several precise steps designed to preserve biological samples in a near-native state and integrate data from different imaging modalities. The process begins with cryo-fixation, which is rapid freezing, typically by plunging the sample into a cryogen like liquid ethane. This vitrification process preserves cells in a hydrated, ice-crystal-free state, crucial for maintaining structural integrity for both light and electron microscopy.
Following cryo-fixation, the frozen sample is first imaged using a cryo-fluorescence light microscope. This initial imaging step allows researchers to identify specific areas of interest, such as a particular organelle, a protein complex, or a cell interacting with a pathogen, based on fluorescent markers. The cryo-stage on the microscope ensures the sample remains at liquid nitrogen temperatures, minimizing ice contamination and maintaining its vitrified state during imaging.
The identified region of interest is then precisely prepared for electron microscopy while remaining frozen. For thicker samples, focused ion beam (FIB) milling creates a thin lamella, typically around 200 nanometers thick, suitable for electron transmission. This targeted thinning ensures that electrons can pass through the sample to generate high-resolution images.
The prepared cryo-sample is then transferred to a cryo-electron microscope, such as a cryo-electron tomograph. Here, ultra-high-resolution images of the targeted area are acquired, revealing its detailed ultrastructure, including individual proteins, membranes, and macromolecular assemblies. Cryo-EM imaging is performed at extremely low temperatures, well below -150°C, to maintain the sample’s vitrified state.
The final step involves the computational alignment and overlaying of the low-resolution contextual image from the light microscope with the high-resolution structural image from the electron microscope. Specialized software correlates these different datasets, allowing scientists to precisely map the location of fluorescently labeled molecules or cellular events within their detailed ultrastructural environment.
Unlocking Cellular Secrets
Cryo-CLEM has significantly advanced our understanding of cellular biology by enabling unprecedented visualization of complex biological events. The technique allows researchers to capture dynamic cellular processes at a specific moment in time and then examine their intricate details with high resolution. For instance, it has provided insights into how viruses interact with host cells during infection, revealing the precise structural changes and molecular rearrangements that occur. Studies have used Cryo-CLEM to visualize pseudotyped HIV-1 particles entering cells, showing the viral membrane, mature core, and surrounding clathrin cages.
The technique has also been instrumental in understanding disease mechanisms by revealing structural alterations or pathogen interactions at a molecular level within their native cellular context. For example, in studies of mumps virus infection, fluorescently labeled stress granules guided FIB milling, allowing researchers to observe mumps virus nucleocapsids and their interactions with cellular components. This helps pinpoint how diseases affect cellular structures and functions.
Cryo-CLEM excels at visualizing molecular machines and specific molecules within their complex cellular environments, providing a level of contextual detail previously unattainable. Researchers have used it to uncover unexpected forms of cytoskeletal components, such as filamentous actin inside microtubule lumens, through in situ cryo-electron tomography, with cryo-fluorescence guiding the identification of relevant cells. This detailed understanding of cellular processes and disease mechanisms can inform the development of new therapeutic strategies and drug discovery efforts.