Contact X‐ray microscopy of living cells by using LiF crystal as imaging detector

In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single‐shot soft X‐ray contact microscopy is presented. High resolved X‐ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X‐ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid‐state X‐ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X‐ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X‐ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high‐quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light.     

High-resolution water window X-ray imaging of in vivo cells and their products using LiF crystal detectors

High contrast imaging of in vivo Chlorella sorokiniana cells with submicron spatial resolution was obtained with a contact water window X-ray microscopy technique using a point-like, laser-plasma produced, water-window X-ray radiation source, and LiF crystals as detectors. This novel type of X-ray imaging detectors is based on photoluminescence of stable electronic point defects, characterized by high intrinsic resolution. The fluorescence images obtained on LiF crystals exposed in single-shot experiments demonstrate the high sensitivity and dynamic range of this new detector. The powerful performances of LiF crystals allowed us to detect the exudates of Chlorella cells in their living medium and their spatial distribution in situ, without any special sample preparation.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

The application of X-ray microscopy in materials science

X-ray microscopy is a form of high resolution radiography that uses low-energy X-rays (≤10 keV) to enhance the contrast between light elements such as hydrogen, carbon, nitrogen and oxygen. As performed on compact laboratory equipment the technique can achieve spatial resolutions of roughly 1 μm in virtually any material given that the specimen is sufficiently thin (typically 0·2–2 mm) to be adequately transparent to low-energy X-rays. Notwithstanding that the technique has lately found favour in biomedical radiology, its considerable potential in other fields, notably the materials sciences, remains largely unexploited. The scope and potential of laboratory X-ray microscopy in the materials sciences is demonstrated here by its application to ceramics, elastomers, coal-chars and reinforced composites. In all cases the technique provided valuable microstructural characterization often unobtainable by any other non-destructive method. These examples demonstrate that laboratory X-ray microscopy offers much to the materials sciences and deserves a wider application than is current.     

High resolution protein localization using soft X-ray microscopy

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive 'water window', where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.     

Non-destructive and quantitative imaging of a nano-structured microchip by ptychographic hard X-ray scanning microscopy

We used hard X-ray scanning microscopy with ptychographic coherent diffraction contrast to image a front-end processed passivated microchip fabricated in 80 nm technology. No sample preparation was needed to image buried interconnects and contact layers with a spatial resolution of slightly better than 40 nm. The phase shift in the sample is obtained quantitatively. With the additional knowledge of the elemental composition determined in parallel by X-ray fluorescence mapping, quantitative information about specific nanostructures is obtained. A significant enhancement in signal-to-noise ratio and spatial resolution is achieved compared to conventional hard X-ray scanning microscopy.     

Scanning transmission X-ray microscopy with a fast framing pixel detector

Scanning transmission X-ray microscopy (STXM) is a powerful imaging technique, in which a small X-ray probe is raster scanned across a specimen. Complete knowledge of the complex-valued transmission function of the specimen can be gained using detection schemes whose every-day use, however, is often hindered by the need of specialized configured detectors or by slow or noisy readout of area detectors. We report on sub-50 nm-resolution STXM studies in the hard X-ray regime using the PILATUS, a fully pixelated fast framing detector operated in single-photon counting mode. We demonstrate a range of imaging modes, including phase contrast and dark-field imaging.     

X-ray imaging of various biological samples using a phase-contrast hard X-ray microscope

In this study, we visualized the internal structures of various bio-samples and found the optimum conditions of test samples for the 7 keV hard X-ray microscope of the Pohang light source. From the captured X-ray images, we could observe the intercellular and intracellular structures of dehydrated human cells and mouse tumor tissues without using any staining materials in a spatial resolution better than 100 nm. The metastasized lung tissue, which was several tens of micrometers in thickness, was found to be very well suited to this hard X-ray microscope system, because it is nearly impossible to observe such a nontransparent and thick sample with a high spatial resolution better than 100 nm using any microscopes such as a soft X-ray microscope, an optical microscope, or an electron microscope.     

Sialolith characterization by scanning electron microscopy and X-ray photoelectron spectroscopy

The objective of this study has been to characterize sialolith, a calcium phosphate deposit that develops in the human oral cavity, by high-resolution field emission scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The morphological and chemical data obtained helped in the determination of their formation mechanism in salivary glands. Sialoliths in the submandibular salivary glands may arise secondary to sialodenitis, but not via a luminal organic nidus. We believe this is the first study that characterizes a sialolith by XPS.     

Read-out of soft X-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.     

Image scanning fluorescence emission difference microscopy based on a detector array

We propose a novel imaging method that enables the enhancement of three‐dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications.     

Phase-contrast hard X-ray microscopy using synchrotron radiation for the diagnosis of onychomycosis

Onychomycosis, or fungal infection of the nail, is a disease seen frequently in clinical settings. However, the rates of positive identification using potassium hydroxide preparations or fungal cultures are relatively low. Precise diagnosis is possible via histopathologic examination to monitor the existence of fungus and performance of a fungal culture for confirmation. Phase-contrast hard X-ray microscopy using synchrotron radiation provides 70-nm spatial resolution and enables imaging of minute internal cellular structures. This study confirms the feasibility of diagnosing onychomycosis using a phase-contrast hard X-ray microscope developed at 1B2 beam line using a Pohang light source.     

Ultrastructural characterization of isolated rat Kupffer cells by transmission X-ray microscopy

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X-ray microscopy as well as transmission electron microscopy. X-ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent-shaped vacuoles that were half X-ray lucent, half X-ray dense; others were uniformly dense. The number of crescent-shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent-shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X-ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent-shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects — crescent-shaped versus uniform vacuoles — might be due to different three-dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron-lucent and electron-dense. Based on the observation of only one type of vacuole by transmission X-ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent-shaped vacuole. From the present data it can be concluded that transmission X-ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of data obtained by transmission electron microscopy.     

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography

Using a cryo scanning transmission X-ray microscope ( Maser, et al . (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc . 197, 68–79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.     

Qualitative detection of Mg content in a leaf of Hedera helix by using X-ray radiation from a laser plasma source

In this article, a method to reveal the presence of Mg content inside the different parts of leaves of Hedera helix is presented. In fact a sample of a Hedera helix's leaf, commonly characterized by a green and a white side, is analyzed under X-ray radiation. The presence of two zones with different colors in the Hedera helix's leaf has not been explained. In this connection, there are presently three hypotheses to explain the characteristic double-color appearance of the leaf. The first hypothesis suggests a different cytoplasmic inheritance of chloroplasts at the cell division, the second a different allelic composition, homozygote and heterozygote, between the two zones, and finally the third the action of a virus which changes the color properties in the Hedera's leaves. The resulting effect is a different content of "something" between the green and the white side. We utilized X-ray radiation, obtained from a plasma source with a Mg target, to image Hedera helix leaves and we found that the green side of the leaf is highlighted. We may suppose that the reason why the X-rays from a Mg plasma source, allow us to pick up the green side is probably due to the greater presence of the amount of Mg (from chlorophyll or other complexes and/or salts) in the two sides, green and white, of the leaf.     

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy

We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 μm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 10¹⁰ Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography ( Wang et al. (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc . 197, 80–93) or spectroscopic analysis.     

Monitoring of the heavy-metal hyperaccumulation in vegetal tissues by X-ray radiography and by femto-second laser induced breakdown spectroscopy

This article reports on the utilization of X-ray microradiography and laser induced breakdown spectroscopy (LIBS) techniques for investigation of the metal accumulation in different part of leaf samples. The potential of the LIBS-analysis for finding the proper plant species for phytoremediation is compared with the results of microradiography measurements at the HERCULES source at ENEA, Rome (Italy) and X-ray microradiography experiments at the ELETTRA Synchrotron, Trieste (Italy).     

Automatic tracking of individual migrating cells using low-magnification dark-field microscopy

Many fundamental biological processes, such as the search for food, immunological responses and wound healing, depend on cell migration. Video microscopy allows the magnitude and direction of cell migration to be documented. Here, we present a simple and inexpensive method for simultaneous tracking of hundreds of migrating cells over periods of several days. Low-magnification dark-field microscopy was used to visualize individual cells whereas time-lapse video images were acquired by computer for future analysis. We employed an automated tracking algorithm to identify individual cells on each video image allowing migration paths to be tracked using a nearest neighbour algorithm. To test the method, we followed the time-course of migration of 3T3 fibroblasts, endothelial cells and individual amoeba in the absence of any chemical stimulus gradient. All cell types showed a 'random walk' behaviour in which mean squared displacement in position increased linearly with time. We defined a 'migration coefficient' (D(mig)), analogous to a diffusion coefficient, which gave an estimate of cell migration rate. D(mig) depended on cell type and temperature. When amoebas were made to undergo chemotaxis, the cells no longer followed a random walk but instead moved at a near constant velocity (V(av)) towards the chemotactic stimulus.     

Quantitative X-ray microanalysis of hydrogen peroxide within plant cells

Using quantitative X-ray microanalysis in combination with CeCl3-based cytochemical staining of hydrogen peroxide (H2O2) we have developed a new solution for quantification of H2O2 at the subcellular level. Quantitative X-ray microanalysis of plastic-embedded leaves of Populus euphratica Oliv. showed that the obtained cerium precipitates by CeCl3 staining were the mixture of cerium perhydroxides and cerium phosphate, in which the fractions of CePO4 were: (1) 52-74% in cell walls of fresh leaf segments, and (2) 34-70% in the cytoplasm in 10 mM H2O2-treated leaf segments that were previously freeze-dried. Taking the concentration of cerium phosphate as staining background, we reached the cellular concentration of cerium perhydroxides and the corresponding concentration of H2O2. Results showed that H2O2 was present in the cytoplasm of rehydrated leaf segments (29-58 mM), but in fresh leaves, H2O2 was observed in the walls of all measured cell types (17-74 mM).