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Synchrotron Miniaturization enabling Innovative Laboratory Equipment in soft x-ray tomography.

Periodic Reporting for period 2 - SMILE (Synchrotron Miniaturization enabling Innovative Laboratory Equipment in soft x-ray tomography.)

Reporting period: 2017-11-01 to 2019-02-28

Societal issue being addressed:
Cancer now accounts for a quarter of all deaths in the EU and it has recently overtaken cardiovascular disease as the number one cause of death in men over 50. Despite billions of Euros of investment in cell biology, science has barely scratched the surface in understanding what is happening within a cancerous cell to make it multiply uncontrollably as the disease spreads through the body. Cell shape and the shape of its internal organelles, are seen as important influencers on cell signalling and gene switching that triggers cancer growth. 3D imaging of the internal structure of whole, intact, cells is playing an increasingly important role in helping scientists to understand cancer as it allows them to accurately and non-intrusively measure and monitor organelle shape.

Role of soft x-ray imaging:
The only technology available today that can image through a whole cell, without needing to slice it or stain it, is soft x-ray imaging, which is carried out using a soft x-ray microscope. The problem is that the soft x-ray illumination required for a soft x-ray microscope is only available from electron accelerator facilities, called synchrotrons. Only four of these billion dollar, football stadium-sized facilities exist that have a soft x-ray beamline and only approximately 100 research groups from a target addressable market of 3,000 disease research organisations have been able to get access, having had to queue for up to twelve months to do so.

SiriusXT's Innovation:
SiriusXT’s innovation has been to miniaturize the synchrotron into a small chamber that will that will easily fit on a laboratory bench. This patented source of soft x-ray illumination, when integrated with a microscope similar to that used at the synchrotrons, will give our target addressable market of 3,000 disease research organizations access to this imaging modality in their own labs, at a fraction of the cost of a synchrotron.

Overall Objectives of Project SMILE
i) to demonstrate a reliable miniaturized source, which is capable of producing sufficient soft x-ray illumination to image a whole cell in 3D in under 60 minutes
ii) to build and demonstrate a soft x-ray microscope, using the miniaturized soft x-ray source.
iii) to carry out a Pilot evaluation of the soft x-ray microscope at the Crick Cancer Research Institute in London
Work performed over the 26 months of Project SMILE (01Jan17 to 28Feb19)

Project SMILE sought to develop an existing prototype of a laboratory-scale soft x-ray microscope into a Pilot system that would be sufficiently reliable to be used for customer evaluation and, in so doing, to begin the process of product commercialisation. SiriusXT had been founded one year prior to the start of Project SMILE with the goal of designing, building and commercialising a laboratory scale Soft X-ray Microscope. During the one-year period prior to the start of Project SMILE SiriusXT developed and built the prototype microscope. The goals of project SMILE were, therefore, 100% aligned with SiriusXT's business strategy.

The first major task in the project was to re-engineer the prototype design so that it was mechanically capable of reliably producing high resolution, high contrast, sample images. The goal was to start first with 2D images of 'dry' (non-biological) samples that could be imaged at room temperature and then to progress to 2D images of cryo-frozen biological samples. Tomograms (3D images) would then be produced by combining a series of 2D images of the sample, taken as the sample was progressively turned around a axis of rotation.

The work required to re-engineer the prototype microscope into a reliable, precision, imaging instrument proved to be much greater than had been anticipated as it required multiple iterations of module redesign. The delays in achieving this goal had a ripple-on effect on the other project goals since most of these required a fully functioning microscope to be available.

Notwithstanding this, the progress made through the project was very significant and the images and performance produced demonstrated an imaging resolution comparable with the synchrotron-based microscopes. Furthermore, SiriusXT has been successful in raising additional funding from institutional investors to continue the product development and to complete all the project goals.

The results obtained from the soft x-ray source testing has been shared with the operators of the European synchrotrons and they have been very supportive with advice on how to integrate the microscope optics for best imaging performance. SiriusXT personnel have also worked closely with operators of cryo-imaging laboratories, who have developed expertise in cryo- cell preparation for electron microscopy applications as well as for soft x-ray imaging at the synchrotron beamlines. By October, 2017, we have the capability to screen cryo-prepared grids, populated with cells, on a fluorescence microscope and to then transfer the frozen cells in a custom-designed transfer box to the cryo stage of the microscope. The results of this cryo-cell handling work has been described and published on the SiriusXT’s LinkedIn page and on the Cryo Microscopy LinkedIn Group page, which has over 1,000 members interested in cryo-cell microscopy.
Over the past ten years or more a number of organisations have attempted to design and build a commercial 'bench-top' soft x-ray source, that would be capable of generating sufficient soft x-ray photon illumination to 3D image a biological cell in less than one hour, however, all attempts to date have failed. The soft x-ray illumination source in the SiriusXT Pilot Microscope has been demonstrated to be provide the image resolution required by target customers. The photon flux demonstrated in the Pilot Microscope does not fully meet the targeted one-hour imaging time but it has clearly demonstrated the capability of achieving this goal in the next design iteration. This achievement is clearly beyond the state of the art.

This achievement will allow SiriusXT to build a commercial, bench-top, soft x-ray microscope, fulfilling a well defined market need for such a product. In so doing, SiriusXT will open up soft x-ray imaging as a commercially viable lab-based technique for a target market of over 3,000 disease research and drug discovery organisations. It will be the only imaging modality available to cell imaging laboratories for generating high resolution images of the complete internal structure of whole biological cells, without having to slice or stain the cells. It will provide scientists working on identifying the root causes of diseases, such as cancer, Alzheimer disease, Huntington disease, etc., a unique insight into the internal function of the diseased cell, increasing their knowledge of these diseases and accelerating the development of effective cures for these diseases.
SXT100 Pilot Soft X-ray Microscope