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Abstract: Researchers have developed a new technique for studying cells in their native state. The goal was to obtain high-quality imaging data from cells without the need for sectioning or chemical fixation. The new method avoids any treatment that would disturb cell structure, so that no artefacts (errors) are introduced into the images. To demonstrate this novel correlative microscopy platform’s effectiveness, the team studied the early stages of cell infection by reoviruses. Although the specific viruses have been studied extensively, there is a debate regarding the method of infection. This research focused on the way that the virus escapes from vesicles, a required step for replication. At beamline B24, using a correlative imaging approach by combining soft X-ray tomography with super resolution microscopy in cryogenic conditions, the team tracked the infection mechanism. The results revealed that the virus had already escaped from the host vesicles two hours after infection, with the vesicles preserving their circular shape, suggesting a gentle, pore-based exit mechanism for the virus. Reoviruses are valuable tools that could be engineered to express proteins and have the potential to be used in vaccines. Knowing the infection mechanism will facilitate their handling and manipulation for biomedical purposes. The new imaging platform has also been used to validate anticancer compounds, study cell structure during development and investigate clearance of human pathogenic microorganism by immune host cells. The work is the outcome of a collective effort between Diamond Light Source and research groups and facilities across Europe, including the University of Oxford, Heidelberg University Hospital, the Université de Nantes and CryoCapCell.

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Abstract: Correlation of 3D images acquired on different microscopes can be a daunting prospect even for experienced users. This protocol describes steps for registration of images from soft X-ray absorption contrast imaging and super-resolution fluorescence imaging of hydrated biological materials at cryogenic temperatures. Although it is developed for data generated at synchrotron beamlines that offer the above combination of microscopies, it is applicable to all analogous imaging systems where the same area of a sample is examined using successive non-destructive imaging techniques.

Open Access

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Abstract: Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

Open Access

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Abstract: This protocol illustrates the steps necessary to deposit correlated 3D cryo-imaging data from cryo-structured illumination microscopy and cryo-soft X-ray tomography with the BioStudies and EMPIAR deposition databases of the European Bioinformatics Institute. There is currently a real need for a robust method of data deposition to ensure unhindered access to and independent validation of correlative light and X-ray microscopy data to allow use in further comparative studies, educational activities, and data mining.

Open Access

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Abstract: Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.