Cryo-electron microscopy (cryo-EM) allows for the observation of high resolution structures of biological samples as close to the native state as possible. In contrast to conventional EM methods no chemical fixatives, such as glutaraldehyde or osmiumtetroxide, or contrast enhancing heavy metal stains, such as uranyl acetate, are used. This minimises the risk of fixation and staining artefacts. Instead, samples are rapidly frozen and visualised directly at the electron microscope while being constantly cooled at temperatures below –160 °C. For small samples – protein complexes, liposomes (see Figure 1), viruses, prokaryotic cells, organelles – immersion freezing ("plunge freezing") is the sample preparation method of choice . Using grid plungers such as the Leica EM GP, thin films of samples that are typically 20 to 500 nm thick are prepared and rapidly frozen in cooled cryogens such as ethane at temperatures just above the cryogen’s melting point in order to achieve sample vitrification . Larger samples (most commonly, eukaryotic cells and tissues) are too thick for plunge freezing and can be prepared using high pressure freezing followed by cryo-sectioning.
Fig. 1: Cryo-EM micrographs showing morphological changes in liposomes (LUVs) caused by a mutation in a membrane protein (NEC220). Scale bars: 500 nm (A); 100 nm (B & C). Modified from Bigalke et al., 2014.
One example of a research field that has especially benefitted from the increased resolution of cryo-EM is the study of cilia and flagella. Cilia and flagella are highly conserved, universal motility and sensing organelles that perform many essential tasks in eukaryotes. Ciliary defects in humans cause a variety of conditions and diseases, collectively named ciliopathies. Linking structure to function is essential to understanding how cilia work and why they fail in disease. Sample preparation by cryo-plunge freezing followed by cryo-electron tomography (cryo-ET) – a three-dimensional (3D) cryo-EM technique – followed by image processing (subtomogram averaging) has proven to be a highly successful strategy to gain unprecedented insights into the 3D structure and function of cilia and flagella (Figures 2 and 3). Cryo-ET was introduced to cilia research only in 2006 and has not only provided higher resolution, near-native state, 3D structures, but also dramatically changed our understanding of how cilia function and what goes wrong in diseases. Compared to just a few years ago we now know that microtubules are not hollow, empty tubes, but contain microtubule inner proteins (MIPs ); the nexin link was discovered decades ago and has now been identified as a part of the dynein regulatory complex ; radial spokes have small structural and possibly functional differences [6, 7] and we have our first glimpses into how the tens of thousands of dynein motor molecules are coordinated to generate ciliary motion . Along with these breakthroughs many new questions have been raised and are still waiting to be answered.
Fig. 2: Cryo-ET derived tomographic slices and isosurface renderings of averaged 96 nm repeats – the building block of eukaryotic cilia and flagella. Scale bar: 100 nm. From Heuser et al., 2012.
Fig. 3: Cryo-ET derived isosurface renderings of averaged central apparatus structures from Chlamydomonas (green algae) and Strongylocentrotus (sea urchin) flagella after averaging. Modified from Carbajal-González et al., 2013.
The increased resolution and quality of cryo-EM comes at a price: known problems include radiation sensitivity (due to the absence of chemical fixatives) and low-contrast, noisy images (low signal-to-noise ratio due to lack of heavy metal contrasting reagents). These challenges can usually be overcome by operating the microscope in low dose mode to minimize sample radiation exposure and using image processing (averaging) to combine the information from many images to increase the signal to noise ratio and ultimately the resolution. Recent progress in phase plate technology and especially direct electron detectors has pushed the achievable resolution towards the near-atomic range .
However, while cryo-EM provides high resolution snapshots of an area of interest it is neither suited for scanning of larger areas nor for live imaging. These shortfalls can be overcome by correlative microscopy techniques such as Correlative Light and Electron Microscopy (CLEM). CLEM combines the best of both worlds. Light/fluorescence microscopy is used for live cell imaging of easy to observe markers (such as green fluorescent protein; GFP) in order to identify the right time and area of interest. Then this area is examined further with the high resolution power of the electron microscope (Figure 4). An exciting new and promising CLEM development is the Leica cryo stage, which allows for observation of cryo samples by light/fluorescence microscopy at temperatures below –160 °C. This not only strongly reduces bleaching of fluorophores but also permits the identification of regions of interest (often fluorescently labelled targets) in the frozen sample by light/fluorescent microscopy first. Then the sample (while still being cooled at below –160 °C) can be easily transferred to the electron microscope in order to revisit the areas of interest for high resolution studies by cryo-EM.
Fig. 4: CLEM of high pressure frozen and freeze substituted HeLa cells (using Leica EM HPM100 and Leica EM AFS2): A cell of interest is identified by a fluorescence marker and visualized by DIC (A, B, D) fluorescence microscopy (C) and EM at low (E), medium (F) and high resolution (G, H). Modified from .
Thus far every resolution increase in EM went hand-in-hand with important new biological discoveries regarding structure and function. And there is no indication to suggest that this trend will end anytime soon.
The EM Facility of the Campus Science Support Facilities GmbH (CSF) acknowledges funding from the Austrian Federal Ministry of Science, Research & Economy and the City of Vienna.
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