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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/ C 3 H 6 O 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.

Laboratory and synchrotron X-ray tomography are powerful tools for non-invasive studies of biological samples at micrometric resolution. In particular, the development of phase contrast imaging is enabling the visualization of sample details with a small range of attenuation coefficients, thus allowing in-depth analyses of anatomical and histological structures. Reproductive medicine is starting to profit from these techniques, mainly applied to animal models. This study reports the first imaging of human ovarian tissue where the samples consisted of surgically obtained millimetre fragments, properly fixed, stained with osmium tetroxide and included in epoxydic resin. Samples were imaged by the use of propagation phase contrast synchrotron radiation micro-computed tomography (microCT), obtained at the SYRMEP beamline of Elettra light source (Trieste, Italy), and X-ray absorption microCT at the Theoretical Biology MicroCT Imaging Laboratory in Vienna, Austria. The reconstructed microCT images were compared with the soft X-ray absorption and phase contrast images acquired at the TwinMic beamline of Elettra in order to help with the identification of structures. The resulting images allow the regions of the cortex and medulla of the ovary to be distinguished, identifying early-stage follicles and visualizing the distribution of blood vessels. The study opens to further application of micro-resolved 3D imaging to improve the understanding of human ovary's structure and support diagnostics as well as advances in reproductive technologies.

Radiation damage is an important aspect to be considered when analysing biological samples with X-ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X-ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation-damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X-ray exposure at different doses on ultralene, paraffin and on paraffin-embedded rat tissues were studied using Fourier-transform infrared (FTIR) microspectroscopy and X-ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose-dependent degradation of their vibrational profiles, consistent with radiation-induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose.

Nanotoxicology and nanomedicine investigations often require the probing of nano-objects such as fibres and particles in biological samples and cells, whilst internalization and intracellular destiny are the main issues for in vitro cellular studies. Various high resolution microscopy techniques are well suited for providing this highly sought-after information. However, sample preparation, nanomaterial composition and sectioning challenges make it often difficult to establish whether the fibres or particles have been internalized or they are simply overlaying or underlying the biological matter. In this paper we suggest a novel suitable combination of two different microscopic techniques to reveal in intact cells the uptake of asbestos fibres by mesothelial cells. After exposure to asbestos fibres and fixation, cells were first analysed under the AFM instrument and then imaged under the TwinMic soft X-ray microscope at Elettra Sincrotrone. The suggested approach combines standard soft X-ray microscopy imaging and AFM microscopy, with a common non-invasive sample preparation protocol which drastically reduces the experimental uncertainty and provides a quick and definitive answer to the nanoparticle cellular and tissue uptake.

The recent upgrading of synchrotron radiation (SR) sources has favored, in the last few years, the construction and design of beamlines optimized for the study of cultural heritage materials, which may require ad hoc setups, specific spatial resolutions, and detection limits. In the field of cultural heritage, integrated approaches combining different techniques are often required, even at large facilities, where some beamlines offer the possibility of performing different types of measurements at the same point of analysis, complementing preliminary information usually obtained by conventional laboratory and/or portable in situ methods. An overview of the last ten years of synchrotron applications for the study of pigments is given, with discussion of upstream and downstream challenges to methods and techniques. The possibilities offered by the synchrotron techniques are illustrated by a case study of a particular class of painted ceramics, as an example of different research questions that are solved by a combination of SR-based methods. [ABSTRACT FROM AUTHOR]

The range of materials analysed at Elettra, the Italian synchrotron facility, in the area of cultural heritage (CH) is very broad and goes from inorganic materials such as metals, stone, glass, ceramics and pigments, to organic-based materials such as wood, paper, leather, fabrics and biological samples. Several applications are involved, such as restoration and conservation, analysis of alteration products, identification of manufacturing techniques used by ancient communities, as well as the study of human and animal evolution. Synchrotron radiation offers a large portfolio of analytical techniques and several advantages, such as the unique non-destructive capabilities of most analytical approaches and the possibility of using synergic way more X-ray techniques in the same environment. In this paper, we will describe the Elettra source and the beamlines routinely dedicated to CH experiments, as well as the office designed to facilitate users with little or no experience in the use of large research infrastructures. [ABSTRACT FROM AUTHOR]

Label-free spectromicroscopy methods offer the capability to examine complex cellular phenomena. Electron and X-ray based spectromicroscopy methods, though powerful, have been hard to implement with hydrated objects due to the vacuum incompatibility of the samples and due to the parasitic signals from (or drastic attenuation by) the liquid matrix surrounding the biological object of interest. Similarly, for many techniques that operate at ambient pressure, such as Fourier transform infrared spectromicroscopy (FTIRM), the aqueous environment imposes severe limitations due to the strong absorption of liquid water in the infrared regime. Here we propose a microfabricated multi-compartmental and reusable hydrated sample platform suitable for use with several analytical techniques, which employs the conformal encapsulation of biological specimens by a few layers of atomically thin graphene. Such an electron, X-ray, and infrared transparent, molecularly impermeable and mechanically robust enclosure preserves the hydrated environment around the object for a sufficient time to allow in situ examination of hydrated bio-objects with techniques operating under both ambient and high vacuum conditions. An additional hydration source, provided by hydrogel pads lithographically patterned in the liquid state near/around the specimen and co-encapsulated, has been added to further extend the hydration lifetime. Note that the in-liquid lithographic electron beam-induced gelation procedure allows for addressable capture and immobilization of the biological cells from the solution. Scanning electron microscopy and optical fluorescence microscopy, as well as synchrotron radiation based FTIR and X-ray fluorescence microscopy, have been used to test the applicability of the platform and for its validation with yeast, A549 human carcinoma lung cells and micropatterned gels as biological object phantoms. [ABSTRACT FROM AUTHOR]

Soft X-ray microscopy coupled with low energy X-ray fluorescence is a powerful tool for investigating complex biological systems like cells and tissues. Due to certain characteristics of X-ray sources, sample stage motors, and detectors, the examination of large areas at high resolutions is very time consuming, often confining the analysis only to a restricted number of pre-selected representative regions. Here we propose and demonstrate a compressive sensing method that provides an alternative approach for overcoming such limitations and can be applied to different kinds of samples and other microscopy and analytical techniques. [ABSTRACT FROM AUTHOR]

Synchrotron X-ray µ- and nano-probes are increasingly affirming their relevance in cultural heritage applications, especially in material characterization of tiny and complex micro-samples which are typical from archaeological and artistic artifacts. For such purposes, synchrotron radiation facilities are tailoring and optimizing beamlines and set-ups for CH, taking also advantages from the challenges offered by the third-generation radiation sources. In ancient ceramics studies, relevant information for the identification of production centers and manufacture technology can be obtained in a non-invasive and non-destructive way at the micro-sample level by combining different SR based methods. However, the selection of appropriate beamlines, techniques and set-ups are critical for the success of the experiments. Fine and varnished wares (e.g., Attic and western-Greek colonial products) are an excellent case study for exploring challenges offered by synchrotron X-ray microprobes optimized to collect microchemical and phase-distribution maps. The determination of provenance and/or technological tracers is relevant in correctly classifying productions, often based only on ceramic paste, gloss macroscopic features or style. In addition, when these vessels are preserved in Museums as masterpieces or intact pieces the application of non-invasive approach at the micro sample is strictly required. Well-designed synchrotron µXRF and µXANES mapping experiments are able providing relevant clues for discriminating workshops and exploring technological aspects, which are fundamental in answering the current archaeological questions on varnished Greek or western-Greek colonial products. [ABSTRACT FROM AUTHOR]

The possibility of fast X‐ray fluorescence (XRF) imaging using on‐the‐fly acquisition is very attractive due to its high acquisition speed. The underlying assumption is that there must be a meaningful flux of XRF emitted photons collected by the detectors. On‐the‐fly XRF has already been demonstrated at mid‐to‐high X‐ray energies, both for laboratory and synchrotron‐based instruments. In this manuscript, we report on the capability of DANTE digital pulse processor (DPP) in the soft X‐ray regime. DANTE is a novel compact and low‐power DPP with high energy resolution and high count rate. This work presents preliminary measurements performed at the TwinMic beamline at Elettra synchrotron (Trieste, Italy) addressing the feasibility of on‐the‐fly low energy XRF imaging. [ABSTRACT FROM AUTHOR]

Crucial for the advancement of electrochemical materials science is understanding the lateral variations in the elemental and chemical state of constituents induced by electrochemical reactions at nanoscales. This requires in situ studies to provide observables that contribute to both modeling beyond the phenomenological level and transducing exactly the functionally relevant quantities A range of X-ray coherent diffraction imaging (CDI) approaches has recently been proposed for imaging beyond the diffraction limit with potentially dramatic improvements of time resolution with chemical sensitivity. In this paper we report a selection of ptychography results obtained in situ after successive steps of electrochemically driven growth, complemented with absorption and phase spectroscopy at high lateral resolution. We demonstrate the onset of morphological instability feature formation and correlate the chemical state of Mn with local growth rate controlled by the current density distribution resulting from morphological evolution. [ABSTRACT FROM AUTHOR]