Electron cryo-microscopy (cryo-EM) allows imaging of particles, including protein complexes, viruses and cells, in their native state at resolutions that supersedes those of other imaging techniques. Common artefacts in conventional electron microscopy caused by heavy metal staining and drying or plastic embedding are circumvented by rapid plunge-freezing of samples.
In single particle analysis, three-dimensional density maps can be calculated (reconstructed) by averaging a large number of particle images. This approach is suitable for reconstruction of regular viruses and other macromolecular complexes and allows their visualization approaching atomic resolution. OPIC houses state-of-the-art equipment for cryo-EM. This includes a Titan Krios (Thermo Fisher Scientific) 300-kV transmission electron microscope equipped with Falcon III and K2 Summit/GIF (Gatan) direction electron detectors, in addition to a Glacios (Thermo Fisher Scientific) 200-kV, also equipped with a Falcon III.
Pleomorphic particles such as cells, organelles and irregular viruses can be studied by tomography. The specimen is tilted in the electron microscope and an image is acquired at each tilt. A tomographic reconstruction (tomogram) can then be calculated, allowing visualization of structures at 2-6 nm resolution. Tilt-holders are available for both single- and dual-axis data collection.
Computational sub-volume averaging allows averaging of a large number of volumes, extracted from the three-dimensional tomographic reconstructions. With this method, resolution of 1nm or better can be reached. These methods also allow the detection of viruses and other macromolecular complexes inside the cell. Groups in OPIC are active in software development to achieve these goals.
Using fluorescence microscopy and fluorescently labelled viruses alone or in combination with immunostaining we can characterise the location, distribution and expression levels of host and viral proteins at a particular point in infection.
Live cell imaging enables us to follow the dynamics of the virus infection and to pre-characterise the investigation system. This way we define the best time points for plunge-freezing of the virus-infected cells to allow subsequent analysis of a particular state of interest by cellular cryo electron tomography.
In correlative microscopy, a fluorescence signal of interest is used as a guidance to efficiently locate a relevant event in the electron microscope. Electron microscopy finder grids providing a coordinate system are typically used for this correlation. For cryo fluorescence microscopy we use a setup based on an inverted fluorescence microscope but also explore alternative designs.
Multiple angle light scattering (MALS) is available to study size, molecular mass and aggregation state of protein complexes, viruses and virus-like particles. Fluorescense spectrophotometry allows characterization of fluorescently labelled particles. OPIC houses a Cary Eclipse equipment (Agilent) and sealed cells suitable for fluorescence measurement of pathogenic organisms.
Cell culture for virus production
OPIC houses facilities for mammalian cell culture for virus infection, production and purification. These include class II safety cabinets for cell culture work and virus handling, cell incubators that can be easily decontaminated after virus production, as well as ultracentrifuges for virus concentration and purification.