Cryo-soft X-ray Tomography

Over the last few decades, we have seen a growing interest in the use of high-resolution imaging to study the structures of biological samples. Increased access to synchrotron sources has also driven the development of imaging capabilities and widening use of cryo-soft X-ray tomography across life sciences.

This technique enables scientists to study relatively thick biological samples, without the need for staining or chemical modification; it allows significantly higher dosage of radiation to be used, without damaging the sample, enabling longer exposures and serial imaging.

By using cryo X-ray microscopy, we are able to capture images of a cell's architecture, to a resolution of 25-40 nanometres; the technique can also be used in correlation with other imaging techniques such as electron tomography and fluorescence microscopy to further enhance findings and investigate intricate molecular interactions within whole cells, whilst maintaining them in a near native state.

It allows the scientists to observe biological activities across a range of scales, for diverse applications such as viral replication, bacteria clearance, parasite egress, sporulation, and many more.

Applications of cryo soft X-ray tomography

A recent study at Diamond looks at the life-cycle of malaria, discovering a new step in the stages of infection.

Working with a number of partner organisations - Birkbeck, The Francis Crick Institute and the Centre for Ultrastructural Imaging at King's College London - Diamond's expert scientists used mapping techniques, including cryo soft X-ray tomography, to visualise red blood cells infected with a species of Plasmodium (parasitic microorganisms that are transmitted by mosquitos). The findings may hold the key to new drug development, specifically targeted at preventing egress, a key stage in the cycle of infection. Find out more.

How X-ray tomography works

X-rays pass through a biological sample and get attenuated based on carbon content (carbon-rich biological structures absorb soft x-rays preferentially.) The generated projections are recorded at small angular increments to create a tilt series of images that provide structural information at different orientations. These are then reconstructed into tomograms, the cellular equivalent of CT scans used in hospitals, to produce a 3D image. Other techniques can be used, in correlation with tomography, to zoom in on specific areas of interest and really interrogate the data.

Preparing for cryo soft x-ray tomography

For cryo X-ray tomography to take place at Diamond, samples are grown on flat grids coated with a carbon substrate to stabilise the cells. Fiducial markers are included in the sample to allow accurate and automated reconstructions of the data. The samples are then frozen using liquid nitrogen-cooled liquid ethane and preliminary mapping undertaken to ensure samples are suitable for imaging (for example, they have not been contaminated during plunge freezing and they retain their original form and structure). X-rays are used to generate mosaic images which provide overviews of whole regions and specific areas of interest are identified for data collection.

Tilt series are normally collected in a range of + 65֯ with a step size of 0.5֯ or 1֯, with exposure times generally in the range of a few seconds per projections.

The amount of data generated from X-ray imaging is vast and processing is fully automated, allowing for near real-time data analysis; in-house alignment and reconstruction software is used to pull together the 3D image. Thereafter, a process of segmentation enables the user to classify objects or areas of interest. Once cellular features have been segmented and classified, further analysis can be done to look at size, shape and localisation to gain further biological insight.

The future

Cryo soft X-ray tomography is still an emerging technique for the area of life sciences and on the whole is still only available using synchrotron sources. Over recent years, it has grown in use due to improved accessibility and improved capabilities. It is a very effective technique to visualise the entire structure of cells in a non-destructive way, enabling them to observe whole cells in a near native state. In addition, data can be correlated across imaging techniques.

Related Publications

Cryo-soft X-ray tomography: using soft X-rays to explore the ultrastructure of whole cells

Maria Harkiolaki , Michele C. Darrow , Matthew C. Spink , Ewelina Kosior , Kyle Dent , Elizabeth Duke

Emerging Topics In Life Sciences; DOI: 10.1042/ETLS20170086

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