Development of Lab-based Cryogenic Hard X-ray Microscopy for Soft Biological Materials

This project seeks to develop cryogenic hard X-ray microscopy (imaging) with sub-micron spatial resolution in the laboratory. It will explore a range of soft biological samples, including plant and animal tissues, investigating the contrast enhancement provided by freezing. It will assess the efficacy of different freezing methods (e.g., flash vs. ramped) on crystallisation within samples, taking advantage of the increased field of view achievable compared with electron microscopy. Furthermore, since hard X-rays possess greater penetrating power, they enable regions of interest within larger biological volumes to be examined.

The biological community considers X-ray photon energies above 6 keV as 'hard'. Lab-based imaging systems typically produce X-rays above 20 keV. Relying upon absorption contrast to differentiate between structures for quantifying morphology, it remains challenging to characterise soft biological materials due to their lower X-ray absorption coefficient. Utilising phase differences generated by X-rays interacting with samples is possible, however, the resulting refractive indexes are very small (compared with optical photons) and it's currently unrealistic to achieve quantitative phase contrast X-ray imaging using laboratory systems, due to incoherent sources and insufficient distance between sample and X-ray detector for phase fringes to propagate for adequate measurement.

Chemical staining is the standard method for providing additional absorption contrast of soft biological materials. Lugol's iodine and heavy metals (osmium, cobalt) are commonly used. Still, for soft biological tissues long X-ray exposure times are required to generate sufficient contrast in labs, and during these extended scan times soft biological materials can degrade. The stains themselves can also induce shrinkage and warping in tissues.

Lab-based hard cryogenic X-ray imaging has synergy with volume electron microscopy, such as focused ion beam SEM, that can produce higher resolution with smaller fields of view, and lab-based soft cryogenic X-ray microscopy, that's currently in development for imaging cells. Correlative workflows using these techniques would allow rich multi-scale volumetric datasets to be obtained.

This project requests funding for a PDRA to explore a range of biological soft materials using a novel cryogenic hard X-ray imaging set-up located within the Future Technology Centre at the University of Portsmouth that is capable of providing sub-micron resolution. It will provide researchers and industrialists access to a method within a lab setting, removing an existing bottleneck created by synchrotron access models.