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In this review we propose to address the question: for the life-science researcher, what does X-ray microscopy have to offer that is not otherwise easily available?
We will see that the answer depends on a combination of resolution, penetrating power, analytical sensitivity, compatibility with wet specimens, and the ease of image interpretation.
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Context 1
... and decalciication are involved in a large variety of biological phenomena in development, aging, normal physiology and disease. STXM ooers a convenient way to measure the distribution and concentration of calcium in tissue sections, cell cultures, etc. The absorption spectrum of calciied tissue Fig. 36 shows a prominent resonance structure near the calcium L absorption edges around 3.5 ...
Citations
... By exploiting the natural contrast of carbon against oxygen, soft X-ray transmission microscopy has a potential for visualizing internal structures of living cells in aqueous environment without the need for staining or labeling that may distort the structure and functionality of cells. However, the radiation damage inflicted on the samples, which may destroy the structures of living cells, has hindered the use of soft X-ray microscopes in studies of living cells [15,16]. ...
Soft X-ray transmission microscopy is a powerful tool for visualizing cellular structures due to the natural contrast between organic material and water, but radiation damage has hindered its application to living cells. We have developed a soft X-ray microscope using femtosecond pulse illumination generated by a soft X-ray free electron laser, with which structural change of cells caused by radiation damage is negligible. Employing Wolter mirrors for illumination and objective optics allowed us to perform soft X-ray imaging with a large field of view, enabling observation of mammalian cells. We successfully captured images of living cells in a culture medium visualizing their carbon distribution. The broad wavelength-tunability of soft X-ray free electron lasers, in conjunction with the achromaticity of Wolter mirrors, enabled wavelength resolved cellular imaging.
... SXT captures cellular substructures in their native state, with rapid data collection (<10 min per cell), allowing analysis of relatively large cell populations [16]. This imaging modality operates within the "water window" energy range [17,18], where carbon-rich materials such as membranes and organelles exhibit higher contrast due to stronger X-ray attenuation compared with the cytosol. This quantitative absorption measure, known as linear absorption coefficient (LAC), facilitates the identification of organelles based on their molecular density and composition [19,20]. ...
The dysfunction of α and β cells in pancreatic islets can lead to diabetes. Many questions remain on the subcellular organization of islet cells during the progression of disease. Existing three-dimensional cellular mapping approaches face challenges such as time-intensive sample sectioning and subjective cellular identification. To address these challenges, we have developed a subcellular feature-based classification approach, which allows us to identify α and β cells and quantify their subcellular structural characteristics using soft X-ray tomography (SXT). We observed significant differences in whole-cell morphological and organelle statistics between the two cell types. Additionally, we characterize subtle biophysical differences between individual insulin and glucagon vesicles by analyzing vesicle size and molecular density distributions, which were not previously possible using other methods. These sub-vesicular parameters enable us to predict cell types systematically using supervised machine learning. We also visualize distinct vesicle and cell subtypes using Uniform Manifold Approximation and Projection (UMAP) embeddings, which provides us with an innovative approach to explore structural heterogeneity in islet cells. This methodology presents an innovative approach for tracking biologically meaningful heterogeneity in cells that can be applied to any cellular system.
... With hydrated algae Chlorella, doses of 0.0015 MGy changed the ultrastructural organization. With hydrated living Chinese hamster ovarian (CHO) cells, doses of about 0.1 MGy caused morphological changes 44 . Dehydrated cells are more resistant to X-ray radiation damage than hydrated cells. ...
... Dehydrated cells are more resistant to X-ray radiation damage than hydrated cells. It has been reported in several studies that dose damage is more visible in wet samples than in dehydrated samples 44,45 . For chemically fixed hydrated samples, doses of about 1 MGy were shown to cause cell shrinkage and mass loss 46 . ...
X-ray computed tomography (XCT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray computed tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XCT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XCT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.
... The X-ray beam damage was investigated at the CARNAÚBA X-ray Nanoprobe beamline of the LNLS/CNPEM [22] . The Sirius storage ring was running at 3 GeV and 100 mA [32] , with the beamline delivering a nanobeam of 200 nm(V) × 500 nm(H) and a photon flux of 5 × 109 ph/s at 10 keV. The methodology to collect information on damage consisted of irradiating a small portion of the sample (5 × 5 µm 2 ) with controlled X-ray doses and environmental conditions, then mapping the previously irradiated area employing reduced X-ray doses over a larger area (10 × 10 µm 2 ). ...
Metal halide perovskites (MHP) suffer from photo-structural-chemical instabilities whose intricacy requires state-of-the-art tools to investigate their properties under various conditions. This study addresses the damage caused by focused X-ray beams on MHP through a correlative multi-technique approach. The damage after high-dose irradiation is noticeable in many ways: the loss of iodine and organic components, whose relative amount is reduced; the formation of an excavated area modifying the sample morphology; and an altered optical reflectivity indicating an optically inactive layer. The damage mechanism combines radiolysis and sputtering processes. Interestingly, the bulk underneath the excavated area maintains the initial halide proportion demonstrated by a stable photoluminescence emission energy. We also show that controlling the beam dose and environment is an excellent strategy to mitigate the dose harm. Hence, we combined a controlled X-ray dose with an inert N2 atmosphere to certify the conditions to probe MHP properties while mitigating damage efficiently. Finally, we applied optimized conditions in an X-ray ptychography experiment, reaching a 15-nm spatial resolution, an outcome that has never been attained in this class of materials.
... Scanning techniques based on the serial acquisition of signals have the fundamental advantage of collecting signal point-by-point in a raster scan, which enables a researcher to simultaneously provide multi-signal information from specific illuminated areas, when multiple types of detectors are installed [1][2][3]. The obtained spatial resolution depends only on the spot size and the step size. ...
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.
... X -RAY inspection has been applied in many different areas for more than a century: in medicine, metallurgy, crystallography, electronics, or environmental science [1], [2], [3], [4], [5]. However, many research groups are still active in this field. ...
This paper describes a major step towards the development of the first fully MEMS X-ray source. It focuses mostly on the development of a transmission target that is responsible for converting the electron beam into radiation. The simulation and experimental results are presented, allowing investigation of the influence of the target material, target thickness, and electron beam energy on the obtained X-ray radiation intensities and spectra. It has been proven that one can apply a silicon membrane as an X-ray target working with energies below 25 keV. It has also been shown that covering the membrane with metals such as nickel or tantalum can significantly improve the radiation intensity. Moreover, the article presents how exactly these metals contribute to radiation spectrum, i.e. which gives more coherent beam and which broadband. 2023-0040
... A single hologram is sensitive to noise, since the finest fringes, which determine the resolution, need to be sampled with a sufficiently high CNR for the phase reconstruction. Therefore, the resolution has a strong relation to the number of photons, i.e. dose on the sample (Rudolph et al., 1990;Kirz et al., 1995;Howells et al., 2009;Hagemann & Salditt, 2017;Du et al., 2020). Using a single-photon-counting detector allows for testing the theoretical fluence-resolution predictions, i.e. the number of photons per pixel needed to achieve a certain resolution in an experiment. ...
Full-field X-ray nanoimaging is a widely used tool in a broad range of scientific areas. In particular, for low-absorbing biological or medical samples, phase contrast methods have to be considered. Three well established phase contrast methods at the nanoscale are transmission X-ray microscopy with Zernike phase contrast, near-field holography and near-field ptychography. The high spatial resolution, however, often comes with the drawback of a lower signal-to-noise ratio and significantly longer scan times, compared with microimaging. In order to tackle these challenges a single-photon-counting detector has been implemented at the nanoimaging endstation of the beamline P05 at PETRA III (DESY, Hamburg) operated by Helmholtz-Zentrum Hereon. Thanks to the long sample-to-detector distance available, spatial resolutions of below 100 nm were reached in all three presented nanoimaging techniques. This work shows that a single-photon-counting detector in combination with a long sample-to-detector distance allows one to increase the time resolution for in situ nanoimaging, while keeping a high signal-to-noise level.
... Subsequently, EUV ptychography provides complex transmission images of the specimens with a half-period spatial resolution of 58 nm. Information about the interior material composition of the investigated microorganisms is extracted from these EUV images based on the analysis of the scattering quotient [38]. This scattering quotient micrograph uncovers the composition of the sample averaged along the propagation direction in each pixel of the image. ...
Table-top extreme ultraviolet (EUV) microscopy offers unique opportunities for label-free investigation of biological samples. Here, we demonstrate ptychographic EUV imaging of two dried, unstained model specimens: germlings of a fungus (Aspergillus nidulans), and bacteria (Escherichia coli) cells at 13.5 nm wavelength. We find that the EUV spectral region, which to date has not received much attention for biological imaging, offers sufficient penetration depths for the identification of intracellular features. By implementing a position-correlated ptychography approach, we demonstrate a millimeter-squared field of view enabled by infrared illumination combined with sub-60 nm spatial resolution achieved with EUV illumination on selected regions of interest. The strong element contrast at 13.5 nm wavelength enables the identification of the nanoscale material composition inside the specimens. Our work will advance and facilitate EUV imaging applications and enable further possibilities in life science.
... 18,20 Cells are in the near-native state in that they are cryo-fixed rather than chemically fixed, which reduces the potential for fixation-induced artifacts. [30][31][32][33] In addition, cell structures are visualized based on their inherent contrast rather than preferential binding of chemical stains, ruling out staining artifacts and providing a reproducible natural contrast for whole-cell modeling research. 25,33,34 Unique to SXT, the contrast of structures is quantitative, reflecting information about the molecular composition of each region of the cell. ...
Developing in silico models that accurately reflect a whole, functional cell is an ongoing challenge in biology. Current efforts bring together mathematical models, probabilistic models, visual representations, and data to create a multi‐scale description of cellular processes. A realistic whole‐cell model requires imaging data since it provides spatial constraints and other critical cellular characteristics that are still impossible to obtain by calculation alone. This review introduces Soft X‐ray Tomography (SXT) as a powerful imaging technique to visualize and quantify the mesoscopic (~25 nm spatial scale) organelle landscape in whole cells. SXT generates three‐dimensional reconstructions of cellular ultrastructure and provides a measured structural framework for whole‐cell modeling. Combining SXT with data from disparate technologies at varying spatial resolutions provides further biochemical details and constraints for modeling cellular mechanisms. We conclude, based on the results discussed here, that SXT provides a foundational dataset for a broad spectrum of whole‐cell modeling experiments.
... be always taken into account in the STXM scheme to consider possible beam damage of the sample. 134 In projection microscopy (Fig. 9c), the image of the sample on the detector is obtained without the use of any lens, with the simplest experimental setup oen also scaled down from the synchrotron to laboratory sources. In the particular case in which the detector is placed just aer the sample, the setup is identical to standard radiography and the spatial resolution is directly determined by the detector pixel size. ...
Synchrotron radiation based techniques are powerful tools for battery research and allow probing a wide range of length scales, with different depth sensitivities and spatial/temporal resolutions. Operando experiments enable characterization during functioning of the cell and are thus a precious tool to elucidate the reaction mechanisms taking place. In this perspective, the current state of the art for the most relevant techniques (scattering, spectroscopy, and imaging) is discussed together with the bottlenecks to address, either specific for application in the battery field or more generic. The former includes the improvement of cell designs, multi-modal characterization and development of protocols for automated or at least semi-automated data analysis to quickly process the huge amount of data resulting from operando experiments. Given the recent evolution in these areas, accelerated progress is expected in the years to come, which should in turn foster battery performance improvements.
