Peering deeper into cells with X-rays

Chidinma Okolo works as a beamline scientist on the B24 correlative cryo-imaging beamline at Diamond Light Source. In this article by colleague Dr Kamal Nahas, they discuss her work in how exploring cellular architecture in three dimensions using X-ray imaging creates exciting opportunities to understand cell biology and disease that compensate for the limitations of existing imaging strategies.

Imaging cellular architecture with X-rays has the potential to transform our understanding of the microscopic world. X-rays have long been used to study structures on large and small scales from organ tissues in medical imaging down to protein structure at the atomic scale. Cellular architecture occupies a middle ground between these two goalposts, but it has only been explored with X-rays for over a decade. By viewing a cell’s entire depth in a single image, researchers can study the three-dimensional geometry of subcellular structures, such as assembling viruses and shapeshifting mitochondria. Set apart from scores of two-dimensional microscopy techniques, this technology promises to help us understand the cellular scale with new perspective.

The B24 beamline at the Diamond Light Source is one of the facilities around the world that uses cryo-soft-X-ray tomography to view cells in a different light. Low energy “soft” X-rays illuminate cryogenically preserved cells at different angles to collect three-dimensional data. X-ray imaging allows the shape and organisation of membranes, organelles, and pathogens in the cell to be visualised, but correlation with fluorescence microscopy on the same sample is needed to discriminate between subcellular structures of similar appearance. In pursuit of this goal, we combine X-ray tomography with structured illumination microscopy, allowing fluorescently labelled components in the sample to be identified.

Harnessing X-rays offers a different view

A notable highlight of X-ray tomography is the minimal sample preparation needed. Samples are preserved in solid form by rapid freezing in liquid ethane. This method protects the three-dimensional structure of the cell in a cryogenic state that closely matches physiological conditions. In contrast, electron microscopy—one of the first tools developed to study cellular architecture—requires multiple sample processing steps, such as chemical fixation, sample dehydration, and resin embedding. Each step can introduce artefacts, and imaging cryoprotected samples with X-ray tomography serves as a valuable complement to ensure changes to subcellular structures are biologically meaningful.

Electron-based techniques produce images of cellular structures at unrivalled resolutions of approximately 5 nanometres, small enough to observe ribosomes. Although X-ray tomography cannot detect such small structures and only achieves a resolution of up to 25 nanometres, it stands out for its capacity to image the entire depth of the cell, owing to the ability of X-rays to penetrate through 10-micrometre thick samples. Electron microscopy can only image samples cut into 0.5-micrometre slices and is restricted to a two-dimensional view. In the past two decades, cryo-electron tomography has gained popularity as a strategy for rapidly imaging cellular features without artefacts in three dimensions, but each sample still needs to be carved into a 0.5-micrometer section. By imaging the complete cell volume, X-ray tomography overcomes the limitations of sectioning, including the low odds that rare or short-lived features will be captured in thinned samples. Three-dimensional electron microscopy has been developed to capture the entire cell depth, but the process is time consuming because it requires multiple rounds of sectioning. By contrast, X-ray tomograms can be acquired in 20 minutes, freeing up time to acquire more images.

Unlike electron microscopy, samples for X-ray tomography do not require staining to detect cellular architecture because “soft” X-rays produce natural contrast in the images depending on how well they transmit through different cellular components. The rays readily pass through liquids in the cell, such as the cytoplasm, and reach the camera, registering as bright spots. However, cellular features rich in carbon like membranes absorb the X-rays and prevent them from hitting the camera, leading to dimmer spots that reflect cellular architecture. In addition to providing a three-dimensional view of the cellular landscape, this natural contrast can be harnessed to compare the density of carbon-rich material in the cell.

Shining a light on disease

Recent years have witnessed novel insights in cell and pathogen biology brought about with X-ray tomography. T cells secrete supramolecular attack particles that destroy infected cells, and X-ray tomography helped reveal that these particles are coated in a carbon-rich shell that may help the T cells bundle cytotoxic secretory proteins together. This imaging technique was also used to study several parasites, bacteria, and viruses, helping to understand how pathogens can be therapeutically targeted. Interactions between reovirus and endosomes in infected cells were observed with this technique to determine how it coordinates its exit from the cell, which could help researchers decipher transmission of the virus. Chlamydia bacteria cluster together to form inclusions that are key to their reproduction in infected cells, and X-ray tomography revealed that these inclusions are regulated by the size of bacterial populations they contain. X-ray tomography has also offered insight into the remodelling of organelles that occurs following infections with herpes simplex virus or the virus responsible for the COVID-19 pandemic, aiding our understanding of the cellular response to viral infection.

Imaging the entire cell volume with X-ray tomography provides a means to study the health of cells in response to different stimuli. In fact, X-ray tomography serves as a novel strategy to assess the safety and efficacy of therapeutics that have yet to undergo clinical trials. For example, cells receiving novel vaccine formulations derived from viruses have been monitored to determine if the so-called active virosomes trigger cell death or transform organelles. The technique can also be used to assess anticancer drugs. Observing how cancer cells respond to iridium-based drug candidates helped to narrow focus on the impact of these drugs on mitochondria, which experienced the most overt reactions of any organelle.

X-ray imaging going forward

X-ray tomography is an underexplored and emerging technique that we are steadily advancing. Our continued work will include installing a spectroscopic component in our X-ray microscope, allowing inorganic elements to be distinguished from carbon-rich material in the cell. Such an approach could expand research into the use of metal-based drugs to treat cancer. We have already commissioned a structured illumination microscope to identify fluorescently labelled components in X-ray tomograms, but we have a strategy in place to combine single-molecule localisation fluorescence microscopy with X-ray tomography as well. This could allow the distribution of individual proteins to be pinpointed against the cellular landscape imaged with X-rays. X-ray tomography is uniquely suited to studying cells and pathogens in three dimensions and our goal is to expand access to the technique for basic and translational research.

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