Impact of Sample Preparation Methods on Single-Cell X-ray Microscopy and Light Elemental Analysis Evaluated by Combined Low Energy X-ray Fluorescence, STXM and AFM.

BACKGROUND: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment's success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. METHODS: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. RESULTS: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. CONCLUSION: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy.

Synchrotron Radiation Study of Gain, Noise, and Collection Efficiency of GaAs SAM-APDs with Staircase Structure.

In hard X-ray applications that require high detection efficiency and short response times, such as synchrotron radiation-based Mössbauer absorption spectroscopy and time-resolved fluorescence or photon beam position monitoring, III-V-compound semiconductors, and dedicated alloys offer some advantages over the Si-based technologies traditionally used in solid-state photodetectors. Amongst them, gallium arsenide (GaAs) is one of the most valuable materials thanks to its unique characteristics. At the same time, implementing charge-multiplication mechanisms within the sensor may become of critical importance in cases where the photogenerated signal needs an intrinsic amplification before being acquired by the front-end electronics, such as in the case of a very weak photon flux or when single-photon detection is required. Some GaAs-based avalanche photodiodes (APDs) were grown by a molecular beam epitaxy to fulfill these needs; by means of band gap engineering, we realised devices with separate absorption and multiplication region(s) (SAM), the latter featuring a so-called staircase structure to reduce the multiplication noise. This work reports on the experimental characterisations of gain, noise, and charge collection efficiencies of three series of GaAs APDs featuring different thicknesses of the absorption regions. These devices have been developed to investigate the role of such thicknesses and the presence of traps or defects at the metal-semiconductor interfaces responsible for charge loss, in order to lay the groundwork for the future development of very thick GaAs devices (thicker than 100 µm) for hard X-rays. Several measurements were carried out on such devices with both lasers and synchrotron light sources, inducing photon absorption with X-ray microbeams at variable and controlled depths. In this way, we verified both the role of the thickness of the absorption region in the collection efficiency and the possibility of using the APDs without reaching the punch-through voltage, thus preventing the noise induced by charge multiplication in the absorption region. These devices, with thicknesses suitable for soft X-ray detection, have also shown good characteristics in terms of internal amplification and reduction of multiplication noise, in line with numerical simulations.

Copper Oxide Nanomaterial Fate in Plant Tissue: Nanoscale Impacts on Reproductive Tissues.

A thorough understanding of the implications of chronic low-dose exposure to engineered nanomaterials through the food chain is lacking. The present study aimed to characterize such a response in Cucurbita pepo L. (zucchini) upon exposure to a potential nanoscale fertilizer: copper oxide (CuO) nanoparticles. Zucchini was grown in soil amended with nano-CuO, bulk CuO (100 mg Kg-1), and CuSO4 (320 mg Kg-1) from germination to flowering (60 days). Nano-CuO treatment had no impact on plant morphology or growth nor pollen formation and viability. The uptake of Cu was comparable in the plant tissues under all treatments. RNA-seq analyses on vegetative and reproductive tissues highlighted common and nanoscale-specific components of the response. Mitochondrial and chloroplast functions were uniquely modulated in response to nanomaterial exposure as compared with conventional bulk and salt forms. X-ray absorption spectroscopy showed that the Cu local structure changed upon nano-CuO internalization, suggesting potential nanoparticle biotransformation within the plant tissues. These findings demonstrate the potential positive physiological, cellular, and molecular response related to nano-CuO application as a plant fertilizer, highlighting the differential mechanisms involved in the exposure to Cu in nanoscale, bulk, or salt forms. Nano-CuO uniquely stimulates plant response in a way that can minimize agrochemical inputs to the environment and therefore could be an important strategy in nanoenabled agriculture.

Synchrotron radiation soft X-ray microscopy and low energy X-ray fluorescence to reveal elemental changes in spermatozoa treated with photobiomodulation therapy.

Male infertility is a worldwide clinical issue that increases the number of couples resorting to assisted reproductive technologies (ARTs) to achieve pregnancy. Photobiomodulation therapy (PBMT) is a promising technique that can biostimulate cells and tissues and it is currently successfully employed to enhance the sperm motility in vitro. Nevertheless, its use has been so far restricted to the research field. In the present work, we exploited two PBMT protocols at an 800 nm wavelength on sperm derived from infertile individuals, detecting an increase in sperm motility 1 hour after irradiation. Moreover, in order to add new information about the molecular effect of PBMT, the content of some light elements was evaluated using high resolution X-ray fluorescence imaging. Interestingly, an increase in Na content was detected in the irradiated samples, possibly suggesting a role of this element in sperm motility; indeed, a low Na content was previously correlated with a poor sperm quality, low semen volume, and modest fertilization rate. Amplifying the knowledge of PBMT in the ART field will expedite the translational potentiality of the PBMT use in clinical settings.

µ-XRF Analysis of Trace Elements in Lapis Lazuli-Forming Minerals for a Provenance Study.

This paper presents new developments on the provenance study of lapis lazuli started by our group in 2008: during the years a multi-technique approach has been exploited to obtain minero-petrographic characterization and creation of a database considering only rock samples of known provenance. Since the final aim of the study is to develop a method to analyze archeological findings and artworks made with lapis lazuli in a completely non-invasive way, ion beam analysis techniques were employed to trace the provenance of the raw material used for the production of artifacts. Continuing this goal and focusing the analysis on determination of more significant minero-chemical markers for the provenance study of trace elements in different minerals, the method was extended with the use of micro X-ray fluorescence (µ-XRF), to test the potential of the technique for this application. The analyzes were focused on diopside and pyrite in lapis lazuli samples of known provenance (Afghanistan, Tajikistan, and Siberia). In addition, µ-XRF data were compared with micro proton-induced X-ray emission (µ-PIXE) results to verify the agreement between the two databases and to compare the analytical performance of both techniques for this application.