Attosecond pulse shaping using a seeded free-electron laser.

Attosecond pulses are central to the investigation of valence- and core-electron dynamics on their natural timescales1-3. The reproducible generation and characterization of attosecond waveforms has been demonstrated so far only through the process of high-order harmonic generation4-7. Several methods for shaping attosecond waveforms have been proposed, including the use of metallic filters8,9, multilayer mirrors10 and manipulation of the driving field11. However, none of these approaches allows the flexible manipulation of the temporal characteristics of the attosecond waveforms, and they suffer from the low conversion efficiency of the high-order harmonic generation process. Free-electron lasers, by contrast, deliver femtosecond, extreme-ultraviolet and X-ray pulses with energies ranging from tens of microjoules to a few millijoules12,13. Recent experiments have shown that they can generate subfemtosecond spikes, but with temporal characteristics that change shot-to-shot14-16. Here we report reproducible generation of high-energy (microjoule level) attosecond waveforms using a seeded free-electron laser17. We demonstrate amplitude and phase manipulation of the harmonic components of an attosecond pulse train in combination with an approach for its temporal reconstruction. The results presented here open the way to performing attosecond time-resolved experiments with free-electron lasers.

New achievements in orbital angular momentum beam characterization using a Hartmann wavefront sensor and the Kirkpatrick-Baez active optical system KAOS.

Advances in physics have been significantly driven by state-of-the-art technology, and in photonics and X-ray science this calls for the ability to manipulate the characteristics of optical beams. Orbital angular momentum (OAM) beams hold substantial promise in various domains such as ultra-high-capacity optical communication, rotating body detection, optical tweezers, laser processing, super-resolution imaging etc. Hence, the advancement of OAM beam-generation technology and the enhancement of its technical proficiency and characterization capabilities are of paramount importance. These endeavours will not only facilitate the use of OAM beams in the aforementioned sectors but also extend the scope of applications in diverse fields related to OAM beams. At the FERMI Free-Electron Laser (Trieste, Italy), OAM beams are generated either by tailoring the emission process on the undulator side or, in most cases, by coupling a spiral zone plate (SZP) in tandem with the refocusing Kirkpatrick-Baez active optic system (KAOS). To provide a robust and reproducible workflow to users, a Hartmann wavefront sensor (WFS) is used for both optics tuning and beam characterization. KAOS is capable of delivering both tightly focused and broad spots, with independent control over vertical and horizontal magnification. This study explores a novel non-conventional `near collimation' operational mode aimed at generating beams with OAM that employs the use of a lithographically manufactured SZP to achieve this goal. The article evaluates the mirror's performance through Hartmann wavefront sensing, offers a discussion of data analysis methodologies, and provides a quantitative analysis of these results with ptychographic reconstructions.

An innovative in situ AFM system for a soft X-ray spectromicroscopy synchrotron beamline.

Multimodal imaging and spectroscopy like concurrent scanning transmission X-ray microscopy (STXM) and X-ray fluorescence (XRF) are highly desirable as they allow retrieving complementary information. This paper reports on the design, development, integration and field testing of a novel in situ atomic force microscopy (AFM) instrument for operation under high vacuum in a synchrotron soft X-ray microscopy STXM-XRF end-station. A combination of µXRF and AFM is demonstrated for the first time in the soft X-ray regime, with an outlook for the full XRF-STXM-AFM combination.

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy.

An improved understanding of an ovary's structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin's solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

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.

Femtosecond covariance spectroscopy.

The success of nonlinear optics relies largely on pulse-to-pulse consistency. In contrast, covariance-based techniques used in photoionization electron spectroscopy and mass spectrometry have shown that a wealth of information can be extracted from noise that is lost when averaging multiple measurements. Here, we apply covariance-based detection to nonlinear optical spectroscopy, and show that noise in a femtosecond laser is not necessarily a liability to be mitigated, but can act as a unique and powerful asset. As a proof of principle we apply this approach to the process of stimulated Raman scattering in α-quartz. Our results demonstrate how nonlinear processes in the sample can encode correlations between the spectral components of ultrashort pulses with uncorrelated stochastic fluctuations. This in turn provides richer information compared with the standard nonlinear optics techniques that are based on averages over many repetitions with well-behaved laser pulses. These proof-of-principle results suggest that covariance-based nonlinear spectroscopy will improve the applicability of fs nonlinear spectroscopy in wavelength ranges where stable, transform-limited pulses are not available, such as X-ray free-electron lasers which naturally have spectrally noisy pulses ideally suited for this approach.

Megapixel scanning transmission soft X-ray microscopy imaging coupled with compressive sensing X-ray fluorescence for fast investigation of large biological tissues.

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.

Combined use of AFM and soft X-ray microscopy to reveal fibres' internalization in mesothelial cells.

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.

Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells.

We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm-1 is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm-1, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm-1 is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.

Current status of the TwinMic beamline at Elettra: a soft X-ray transmission and emission microscopy station.

The current status of the TwinMic beamline at Elettra synchrotron light source, that hosts the European twin X-ray microscopy station, is reported. The X-ray source, provided by a short hybrid undulator with source size and divergence intermediate between bending magnets and conventional undulators, is energy-tailored using a collimated plane-grating monochromator. The TwinMic spectromicroscopy experimental station combines scanning and full-field imaging in a single instrument, with contrast modes such as absorption, differential phase, interference and darkfield. The implementation of coherent diffractive imaging modalities and ptychography is ongoing. Typically, scanning transmission X-ray microscopy images are simultaneously collected in transmission and differential phase contrast and can be complemented by chemical and elemental analysis using across-absorption-edge imaging, X-ray absorption near-edge structure or low-energy X-ray fluorescence. The lateral resolutions depend on the particular imaging and contrast mode chosen. The TwinMic range of applications covers diverse research fields such as biology, biochemistry, medicine, pharmacology, environment, geochemistry, food, agriculture and materials science. They will be illustrated in the paper with representative results.

Identification of the primary lesion of toxic aluminum in plant roots.

Despite the rhizotoxicity of aluminum (Al) being identified over 100 years ago, there is still no consensus regarding the mechanisms whereby root elongation rate is initially reduced in the approximately 40% of arable soils worldwide that are acidic. We used high-resolution kinematic analyses, molecular biology, rheology, and advanced imaging techniques to examine soybean (Glycine max) roots exposed to Al. Using this multidisciplinary approach, we have conclusively shown that the primary lesion of Al is apoplastic. In particular, it was found that 75 µm Al reduced root growth after only 5 min (or 30 min at 30 µm Al), with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone. An alteration in the biosynthesis and distribution of ethylene and auxin was a second, slower effect, causing both a transient decrease in the rate of cell elongation after 1.5 h but also a longer term gradual reduction in the length of the elongation zone. These findings show the importance of focusing on traits related to cell wall composition as well as mechanisms involved in wall loosening to overcome the deleterious effects of soluble Al.

Fabrication of a sealed electrochemical microcell for in situ soft X-ray microspectroscopy and testing with in situ co-polypyrrole composite electrodeposition for Pt-free oxygen electrocatalysis.

In this paper we report on the fabrication and testing of a novel concept of sealed electrochemical microcell for in situ soft X-ray microspectroscopy in transmission, dedicated for nonvacuum compatible electrolytes. The microcell, fabricated using ultraviolet lithography, at variance with previous versions of electrochemical wet cells, that featured an optical window glued on top of the electrode system and a very limited electrolyte volume, the device presented here is a single solid block based around a microfabricated channel with fixed optical windows and apt for microfluidic work. Moreover, this cell allows to employ an advanced electrodic geometry developed in our group - so far used only in open electrochemical cells for work with vacuum-compatible electrolytes - also with low-vapor pressure liquids, possibly saturated with the required gases. The cell optimal electrode design allows three-electrode electrochemical control typical of traditional electrochemical experiments. The first electrochemical experiments with this new cell explore the electrochemical growth of a Co-polypyrrole, a composite electrocatalyst material with promising performance to replace the expensive Pt catalyst in fuel-cell oxygen electrodes. Morphological and chemical-state distributions of Co codeposited with polypyrrole has been followed as a function of time and position, yielding unprecedented information on the processes relevant to the synthesis of this catalyst.

The effect of near-infrared Photobiomodulation therapy on the ion content of 50B11 sensory neurons measured through XRF analysis.

Photobiomodulation therapy (PBMT) is a form of treatment commonly used for routine clinical applications, such as wound healing of the skin and reduction of inflammation. Additionally, PBMT has been explored for its potential in pain relief. In this work, we investigated the effect of PBMT on ion content within the 50B11 sensory neurons cell line in vitro using X-Ray fluorescence (XRF) and atomic force microscope (AFM) analysis. Two irradiation protocols were selected utilizing near-infrared laser lights at 800 and 970 nm, with cell fixation immediately following irradiation. Results showed a decrease in Calcium content after irradiation with both protocols, and with lidocaine, used as an analgesic control. Furthermore, a reduction in Potassium content was observed, particularly evident when normalized to cellular volume. These findings provide valuable insights into the molecular impact of PBMT within 50B11 sensory neurons under normal conditions. Such understanding may contribute to the wider adoption of PBMT as a therapeutic approach.

Feasibility of direct mapping of cerebral fluorodeoxy-D-glucose metabolism in situ at subcellular resolution using soft X-ray fluorescence.

Glucose metabolism is difficult to image with cellular resolution in mammalian brain tissue, particularly with (18) fluorodeoxy-D-glucose (FDG) positron emission tomography (PET). To this end, we explored the potential of synchrotron-based low-energy X-ray fluorescence (LEXRF) to image the stable isotope of fluorine (F) in phosphorylated FDG (DG-6P) at 1 µm(2) spatial resolution in 3-µm-thick brain slices. The excitation-dependent fluorescence F signal at 676 eV varied linearly with FDG concentration between 0.5 and 10 mM, whereas the endogenous background F signal was undetectable in brain. To validate LEXRF mapping of fluorine, FDG was administered in vitro and in vivo, and the fluorine LEXRF signal from intracellular trapped FDG-6P over selected brain areas rich in radial glia was spectrally quantitated at 1 µm(2) resolution. The subsequent generation of spatial LEXRF maps of F reproduced the expected localization and gradients of glucose metabolism in retinal Müller glia. In addition, FDG uptake was localized to periventricular hypothalamic tanycytes, whose morphological features were imaged simultaneously by X-ray absorption. We conclude that the high specificity of photon emission from F and its spatial mapping at ≤1 µm resolution demonstrates the ability to identify glucose uptake at subcellular resolution and holds remarkable potential for imaging glucose metabolism in biological tissue.

Advances in sparse dynamic scanning in spectromicroscopy through compressive sensing.

Scanning microscopies and spectroscopies like X-ray Fluorescence (XRF), Scanning Transmission X-ray Microscopy (STXM), and Ptychography are of very high scientific importance as they can be employed in several research fields. Methodology and technology advances aim at analysing larger samples at better resolutions, improved sensitivities and higher acquisition speeds. The frontiers of those advances are in detectors, radiation sources, motors, but also in acquisition and analysis software together with general methodology improvements. We have recently introduced and fully implemented an intelligent scanning methodology based on compressive sensing, on a soft X-ray microscopy beamline. This demonstrated sparse low energy XRF scanning of dynamically chosen regions of interest in combination with STXM, yielding spectroimaging data in the megapixel-range and in shorter timeframes than were previously not feasible. This research has been further developed and has been applied to scientific applications in biology. The developments are mostly in the dynamic triggering decisional mechanism in order to incorporate modern Machine Learning (ML) but also in the suitable integration of the method in the control system, making it available for other beamlines and imaging techniques. On the applications front, the method was previously successfully used on different samples, from lung and ovarian human tissues to plant root sections. This manuscript introduces the latest methodology advances and demonstrates their applications in life and environmental sciences. Lastly, it highlights the auxiliary development of a mobile application, designed to assist the user in the selection of specific regions of interest in an easy way.

Automatic Differentiation for Inverse Problems in X-ray Imaging and Microscopy.

Computational techniques allow breaking the limits of traditional imaging methods, such as time restrictions, resolution, and optics flaws. While simple computational methods can be enough for highly controlled microscope setups or just for previews, an increased level of complexity is instead required for advanced setups, acquisition modalities or where uncertainty is high; the need for complex computational methods clashes with rapid design and execution. In all these cases, Automatic Differentiation, one of the subtopics of Artificial Intelligence, may offer a functional solution, but only if a GPU implementation is available. In this paper, we show how a framework built to solve just one optimisation problem can be employed for many different X-ray imaging inverse problems.

Reconstruction of 3D topographic landscape in soft X-ray fluorescence microscopy through an inverse X-ray-tracing approach based on multiple detectors.

The study of X-ray fluorescence (XRF) emission spectra is a powerful technique used in applications that range from biology to cultural heritage. Key objectives of this technique include identification and quantification of elemental traces composing the analyzed sample. However, precise derivation of elemental concentration is often hampered by self-absorption of the XRF signal emitted by light constituents. This attenuation depends on the amount of sample present between the radiation source and detection system and allows for the exploitation of self-absorption in order to recover a sample topography. In this work, an X-ray-tracing application based on the use of multiple silicon drift detectors, is introduced to inversely reconstruct a 3D sample with correct topographical landscape, from 2D XRF count rates maps obtained from spectroscopy. The reconstruction was tested on the XRF maps of a simulated sample, which is composed of three cells with different size but similar composition. We propose to use the recovered 3D sample topography in order to numerically compute the self-absorption effects on the X-ray fluorescence radiation, thereby showing that a quantitative correction is possible. Lastly, we present a web application which implements the suggested methodology, in order to demonstrate its feasibility and applicability, available at: https://github.com/ElettraSciComp/xrfstir .

PyPore3D: An Open Source Software Tool for Imaging Data Processing and Analysis of Porous and Multiphase Media.

In this work, we propose the software library PyPore3D, an open source solution for data processing of large 3D/4D tomographic data sets. PyPore3D is based on the Pore3D core library, developed thanks to the collaboration between Elettra Sincrotrone (Trieste) and the University of Trieste (Italy). The Pore3D core library is built with a distinction between the User Interface and the backend filtering, segmentation, morphological processing, skeletonisation and analysis functions. The current Pore3D version relies on the closed source IDL framework to call the backend functions and enables simple scripting procedures for streamlined data processing. PyPore3D addresses this limitation by proposing a full open source solution which provides Python wrappers to the the Pore3D C library functions. The PyPore3D library allows the users to fully use the Pore3D Core Library as an open source solution under Python and Jupyter Notebooks PyPore3D is both getting rid of all the intrinsic limitations of licensed platforms (e.g., closed source and export restrictions) and adding, when needed, the flexibility of being able to integrate scientific libraries available for Python (SciPy, TensorFlow, etc.).

A modular software framework for the design and implementation of ptychography algorithms.

Computational methods are driving high impact microscopy techniques such as ptychography. However, the design and implementation of new algorithms is often a laborious process, as many parts of the code are written in close-to-the-hardware programming constructs to speed up the reconstruction. In this article, we present SciComPty, a new ptychography software framework aiming at simulating ptychography datasets and testing state-of-the-art and new reconstruction algorithms. Despite its simplicity, the software leverages GPU accelerated processing through the PyTorch CUDA interface. This is essential for designing new methods that can readily be employed. As an example, we present an improved position refinement method based on Adam and a new version of the rPIE algorithm, adapted for partial coherence setups. Results are shown on both synthetic and real datasets. The software is released as open-source.

Compressive Sensing for Dynamic XRF Scanning.

X-Ray Fluorescence (XRF) scanning is a widespread technique of high importance and impact since it provides chemical composition maps crucial for several scientific investigations. There are continuous requirements for larger, faster and highly resolved acquisitions in order to study complex structures. Among the scientific applications that benefit from it, some of them, such as wide scale brain imaging, are prohibitively difficult due to time constraints. However, typically the overall XRF imaging performance is improving through technological progress on XRF detectors and X-ray sources. This paper suggests an additional approach where XRF scanning is performed in a sparse way by skipping specific points or by varying dynamically acquisition time or other scan settings in a conditional manner. This paves the way for Compressive Sensing in XRF scans where data are acquired in a reduced manner allowing for challenging experiments, currently not feasible with the traditional scanning strategies. A series of different compressive sensing strategies for dynamic scans are presented here. A proof of principle experiment was performed at the TwinMic beamline of Elettra synchrotron. The outcome demonstrates the potential of Compressive Sensing for dynamic scans, suggesting its use in challenging scientific experiments while proposing a technical solution for beamline acquisition software.