Application of a complex constraint for biological samples in coherent diffractive imaging.

We demonstrate the application of a complex constraint in the reconstruction of images from phase-diverse Fresnel coherent diffraction data for heterogeneous biological objects. The application of this constraint is shown to improve the quality of the reconstruction of both the phase and the magnitude of the complex object transmission function.

Phase-diverse Fresnel coherent diffractive imaging of malaria parasite-infected red blood cells in the water window.

Phase-diverse Fresnel coherent diffractive imaging has been shown to reveal the structure and composition of biological specimens with high sensitivity at nanoscale resolution. However, the method has yet to be applied using X-ray illumination with energy in the so-called 'water-window' that lies between the carbon and oxygen K edges. In this range, differences in the strength of the X-ray interaction for protein based biological materials and water is increased. Here we demonstrate a proof-of-principle application of FCDI at an X-ray energy within the water-window to a dehydrated cellular sample composed of red blood cells infected with the trophozoite stage of the malaria parasite, Plasmodium falciparum. Comparison of the results to both optical and electron microscopy shows that the correlative imaging methods that include water-window FCDI will find utility in studying cellular architecture.

An olivine cumulate outcrop on the floor of Jezero crater, Mars.

The geological units on the floor of Jezero crater, Mars, are part of a wider regional stratigraphy of olivine-rich rocks, which extends well beyond the crater. We investigated the petrology of olivine and carbonate-bearing rocks of the Séítah formation in the floor of Jezero. Using multispectral images and x-ray fluorescence data, acquired by the Perseverance rover, we performed a petrographic analysis of the Bastide and Brac outcrops within this unit. We found that these outcrops are composed of igneous rock, moderately altered by aqueous fluid. The igneous rocks are mainly made of coarse-grained olivine, similar to some martian meteorites. We interpret them as an olivine cumulate, formed by settling and enrichment of olivine through multistage cooling of a thick magma body.

Elemental characterisation of the pyramidal neuron layer within the rat and mouse hippocampus.

A unique combination of sensitivity, resolution, and penetration make X-ray fluorescence imaging (XFI) ideally suited to investigate trace elemental distributions in the biological context. XFI has gained widespread use as an analytical technique in the biological sciences, and in particular enables exciting new avenues of research in the field of neuroscience. In this study, elemental mapping by XFI was applied to characterise the elemental content within neuronal cell layers of hippocampal sub-regions of mice and rats. Although classical histochemical methods for metal detection exist, such approaches are typically limited to qualitative analysis. Specifically, histochemical methods are not uniformly sensitive to all chemical forms of a metal, often displaying variable sensitivity to specific "pools" or chemical forms of a metal. In addition, histochemical methods require fixation and extensive chemical treatment of samples, creating the strong likelihood for metal redistribution, leaching, or contamination. Direct quantitative elemental mapping of total elemental pools, in situ within ex vivo tissue sections, without the need for chemical fixation or addition of staining reagents is not possible with traditional histochemical methods; however, such a capability, which is provided by XFI, can offer an enormous analytical advantage. The results we report herein demonstrate the analytical advantage of XFI elemental mapping for direct, label-free metal quantification, in situ within ex vivo brain tissue sections. Specifically, we definitively characterise for the first time, the abundance of Fe within the pyramidal cell layers of the hippocampus. Localisation of Fe to this cell layer is not reproducibly achieved with classical Perls histochemical Fe stains. The ability of XFI to directly quantify neuronal elemental (P, S, Cl, K, Ca, Fe, Cu, Zn) distributions, revealed unique profiles of Fe and Zn within anatomical sub-regions of the hippocampus i.e., cornu ammonis 1, 2 or 3 (CA1, CA2 or CA3) sub-regions. Interestingly, our study reveals a unique Fe gradient across neuron populations within the non-degenerating and pathology free rat hippocampus, which curiously mirrors the pattern of region-specific vulnerability of the hippocampus that has previously been established to occur in various neurodegenerative diseases.

Molar concentration from sequential 2-D water-window X-ray ptychography and X-ray fluorescence in hydrated cells.

Recent developments in biological X-ray microscopy have allowed structural information and elemental distribution to be simultaneously obtained by combining X-ray ptychography and X-ray fluorescence microscopy. Experimentally, these methods can be performed simultaneously; however, the optimal conditions for each measurement may not be compatible. Here, we combine two distinct measurements of ultrastructure and elemental distribution, with each measurement performed under optimised conditions. By combining optimised ptychography and fluorescence information we are able to determine molar concentrations from two-dimensional images, allowing an investigation into the interactions between the environment sensing filopodia in fibroblasts and extracellular calcium. Furthermore, the biological ptychography results we present illustrate a point of maturity where the technique can be applied to solve significant problems in structural biology.