Quantitative imaging of Candida utilis and its organelles by soft X‐ray Nano‐CT

Soft X‐ray microscopy has excellent characteristics for imaging cells and subcellular structures. In this paper, the yeast strain, Candida utilis, was imaged by soft X‐ray microscopy and three‐dimensional volumes were reconstructed with the SART‐TV method. We performed segmentation on the reconstruction in three dimensions and identified several types of subcellular architecture within the specimen cells based on their linear absorption coefficient (LAC) values. Organelles can be identified by the correlation between the soft X‐ray LAC values and the subcellular architectures. Quantitative analyses of the volume ratio of organelles to whole cell in different phases were also carried out according to the three‐dimensional datasets. With such excellent features, soft X‐ray imaging has a great influence in the field of biological cellular and subcellular research.     

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.     

Comparative analysis of isolated cellular organelles by means of soft X-ray contact microscopy with laser-plasma source and transmission electron microscopy

Soft X‐ray contact microscopy (SXCM) is, at present, a useful tool for the examination at submicrometre resolution of biological systems maintained in their natural hydrated conditions. Among current X‐ray‐generating devices, laser‐plasma sources are now easily available and, owing to their pulse nature, offer the opportunity to observe living biological samples before radiation damage occurs, even if the resolution achievable is not as high as with synchrotron‐produced X‐rays. To assess the potential of laser‐plasma source SXCM in the study of cellular organelles, we applied it for the analysis of chloroplasts extracted from spinach leaves and mitochondria isolated from bovine heart and liver. X‐ray radiation was generated by a nanosecond laser‐plasma source, produced by a single shot excimer XeCl laser focused onto an yttrium target. The images obtained with SXCM were then compared with those produced by transmission electron microscopy observation of the same samples prepared with negative staining, a technique requiring no chemical fixation, in order to facilitate their interpretation and test the applicability of SXCM imaging.     

Small-scale in vivo imaging of soft tissues by radioscopy using single X-ray photon counting

A method has been developed for routine laboratory visualisation of small‐scale soft tissue by means of transmission X‐ray radioscopy and tomography. Using termites as models, imaging quality with a spatial resolution of about 3 μm was achieved and 3D tomographic reconstruction was demonstrated. A termite worker individual was visualized before and after its metamorphosis towards the soldier caste. The developed methodology represents a non‐invasive and real‐time way of acquiring 3D anatomic data with a high contrast so that it is a promising candidate to become a tool for routine investigations in life sciences.     

Synchrotron nanoscopy imaging study of scalp hair in breast cancer patients and healthy individuals: Difference in medulla loss and cortical membrane enhancements

Nanoscopic synchrotron X‐ray imaging was performed on scalp hair samples of patients with breast cancer and healthy individuals to investigate any structural differences as diagnostic tool. Hair strands were divided into 2‐3 segments along the strands from root to tip, followed by imaging either in projection or in CT scanning with a monochromatic 6.78‐keV X‐ray using zone‐plate optics with a resolving power of 60 nm. All the examined cancer hairs exhibited medulla loss with cancer stage‐dependent pattern; complete loss, discontinuous or trace along the strands. In contrast, medullas were well retained without complete loss in the healthy hair. In the CT‐scanned axial images, the cortical spindle compartments had no contrast in the healthy hair, but appeared hypointense in contrast to the surrounding hyperintense cortical membrane complex in the cancer hair. In conclusion, observation of medulla loss and cortical membrane enhancements in the hair strands of breast cancer patients demonstrated structural variations in the cancer hair, providing a new platform for further synchrotron X‐ray imaging study of screening breast cancer patients. Microsc. Res. Tech. 79:23–30, 2016. © 2015 Wiley Periodicals, Inc.     

Imaging of cochlear tissue with a grating interferometer and hard X‐rays

This article addresses an important current development in medical and biological imaging: the possibility of imaging soft tissue at resolutions in the micron range using hard X‐rays. Challenging environments, including the cochlea, require the imaging of soft tissue structure surrounded by bone. We demonstrate that cochlear soft tissue structures can be imaged with hard X‐ray phase contrast. Furthermore, we show that only a thin slice of the tissue is required to introduce a large phase shift. It is likely that the phase contrast image of the soft tissue structures is sufficient to image the structures even if surrounded by bone. For the present set of experiments, structures with low‐absorption contrast have been visualized using in‐line phase contrast imaging and a grating interferometer. The experiments have been performed at the Advanced Photon Source at Argonne National Laboratories, a third generation source of synchrotron radiation. The source provides highly coherent X‐ray radiation with high‐photon flux (>10¹² photons/s) at high‐photon energies (5–70 keV). Radiographic and light microscopy images of the gerbil cochlear slice samples were compared. It has been determined that a 20‐μm thick tissue slice induces a phase shift between 1/3π and 2/3π. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Three‐dimensional visualization of rat retina by X‐ray differential phase contrast tomographic microscopy

The retina is one of the most tiny and sophisticated tissues of the body. Three dimensional (3D) visualization of the whole retina is valuable both in clinical and research arenas. The tissue has been predominantly assessed by time‐consuming histopathology and optical coherence tomography (OCT) in research and clinical arenas. However, none of the two methods can provide 3D imaging of the retina. The purpose of this study is to give a volumetric visualization of rat retina at submicron resolution, using an emerging imaging technique‐phase‐contrast X‐ray CT. A Sprague‐Dawley (SD) rat eye specimen was scanned with X‐ray differential phase contrast tomographic microscopy (DPC‐microCT) equipped at the Swiss Light Source synchrotron. After scanning, the specimen was subjected to routine histology procedures and severed as a reference. The morphological characteristics and signal features of the retina in the DPC‐microCT images were evaluated. The total retina and its sublayers thicknesses were measured on the DPC‐microCT images and compared with those obtained from the histological sections. The retina structures revealed by DPC‐microCT were highly consistent with the histological section. In this study, we achieved nondestructive 3D visualization of SD rat retina. In addition to detailed anatomical structures, the objective parameters provided by DPC‐microCT make it a useful tool for retinal research and disease diagnosis in the early stage.     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

A method for estimating spatial resolution of real image in the Fourier domain

Spatial resolution is a fundamental parameter in structural sciences. In crystallography, the resolution is determined from the detection limit of high‐angle diffraction in reciprocal space. In electron microscopy, correlation in the Fourier domain is used for estimating the resolution. In this paper, we report a method for estimating the spatial resolution of real images from a logarithmic intensity plot in the Fourier domain. The logarithmic intensity plots of test images indicated that the full width at half maximum of a Gaussian point spread function can be estimated from the images. The spatial resolution of imaging X‐ray microtomography using Fresnel zone‐plate optics was also estimated with this method. A cross section of a test object visualized with the imaging microtomography indicated that square‐wave patterns up to 120‐nm pitch were resolved. The logarithmic intensity plot was calculated from a tomographic cross section of brain tissue. The full width at half maximum of the point spread function estimated from the plot coincided with the resolution determined from the test object. These results indicated that the logarithmic intensity plot in the Fourier domain provides an alternative measure of the spatial resolution without explicitly defining a noise criterion.     

Evaluation of X‐ray tomography contrast agents: A review of production,protocols, and biological applications

X‐ray computed tomography is a strong tool that finds many applications both in medical applications and in the investigation of biological and nonbiological samples. In the clinics, X‐ray tomography is widely used for diagnostic purposes whose three‐dimensional imaging in high resolution helps physicians to obtain detailed image of investigated regions. Researchers in biological sciences and engineering use X‐ray tomography because it is a nondestructive method to assess the structure of their samples. In both medical and biological applications, visualization of soft tissues and structures requires special treatment, in which special contrast agents are used. In this detailed report, molecule‐based and nanoparticle‐based contrast agents used in biological applications to enhance the image quality were compiled and reported. Special contrast agent applications and protocols to enhance the contrast for the biological applications and works to develop nanoparticle contrast agents to enhance the contrast for targeted drug delivery and general imaging applications were also assessed and listed.     

Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

High resolution SEM of bacterial virus T7.

High resolution "low-loss" scanning electron microscopy is a relatively new technique which permits an investigator to examine structures that were formerly visualized exclusively by transmission electron microscopy [1]. This paper presents some images of intact bacterial virus T7, viewed at the ultrastructural level. Due to the high resolution capibility of this technique, and the demanding physical prerequisites for visualization of the specimen, current specimen preparation techniques were modified in order to permit 1--2 nm resolution in surface mode. Using this method of microscopy, it is possible to view clearly this small bacteriophage (the smallest of the T-coliphages), adsorbed to its host bacterium, in a scanning mode at magnification (and resolution) comparable to TEM without resorting to the use of replicas, or reconstruction of a two-dimensional image.     

An exploratory study of contrast agents for soft tissue visualization by means of high resolution X‐ray computed tomography imaging

High resolution X‐ray computed tomography (CT), or microCT, is a promising and already widely used technique in various scientific fields. Also for histological purposes it has great potential. Although microCT has proven to be a valuable technique for the imaging of bone structures, the visualization of soft tissue structures is still an important challenge due to their low inherent X‐ray contrast. One way to achieve contrast enhancement is to make use of contrast agents. However, contrary to light and electron microscopy, knowledge about contrast agents and staining procedures is limited for X‐ray CT. The purpose of this paper is to identify useful X‐ray contrast agents for soft tissue visualization, which can be applied in a simple way and are also suited for samples larger than (1 cm)³. And 28 chemical substances have been investigated. All chemicals were applied in the form of concentrated aqueous solutions in which the samples were immersed. First, strips of green Bacon were stained to evaluate contrast enhancement between muscle and adipose tissue. Furthermore it was also tested whether the contrast agents remained fixed in the tissue after staining by re‐immersing them in water. Based on the results, 12 contrast agents were selected for further testing on postmortem mice hind legs, containing a variety of different tissues, including muscle, fat, bone, cartilage and tendons. It was evaluated whether the contrast agents allowed a clearer distinction between the different soft tissue structures present. Finally also penetration depth was measured. And 26 chemicals resulted in contrast enhancement between muscle and adipose tissue in the Bacon strips. Mercury(II)chloride (HgCl₂), phosphotungstic acid (PTA), phosphomolybdic acid (PMA) and ammonium orthomolybdate ((NH₄)₂MoO₄) remained fixed after re‐immersion in water. The penetration tests showed that potassium iodide (KI) and sodium tungstate can be most efficiently used for large samples of the order of several tens of cm³. PMA, PTA, HgCl₂ and also to a lesser extent Na₂WO₄ and (NH₄)₂MoO₄ allowed a clearer distinction between the different soft tissue structures present.     

Wavelet-based image restoration for compact X-ray microscopy

Compact water‐window X‐ray microscopy with short exposure times will always be limited on photons owing to sources of limited power in combination with low‐efficency X‐ray optics. Thus, it is important to investigate methods for improving the signal‐to‐noise ratio in the images. We show that a wavelet‐based denoising procedure significantly improves the quality and contrast in compact X‐ray microscopy images. A non‐decimated, discrete wavelet transform (DWT) is applied to original, noisy images. After applying a thresholding procedure to the finest scales of the DWT, by setting to zero all wavelet coefficients of magnitude below a prescribed value, the inverse DWT to the thresholded DWT produces denoised images. It is concluded that the denoising procedure has potential to reduce the exposure time by a factor of 2 without loss of relevant image information.     

Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object

We demonstrate simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object. Subject to the assumptions explicitly stated in the derivation, the algorithm solves the twin‐image problem of in‐line holography and is capable of analysing data obtained using X‐ray microscopy, electron microscopy, neutron microscopy or visible‐light microscopy, especially as they relate to defocus and point projection methods. Our simple, robust, non‐iterative and computationally efficient method is applied to data obtained using an X‐ray phase contrast ultramicroscope.     

Imaging analysis of the interface between osteoblasts and microrough surfaces of laser‐sintered titanium alloy constructs

Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X‐ray spectroscopy; and transmission electron microscopy (TEM) for high‐resolution imaging. FIB was used to prepare thin sections of the cell‐material interface for TEM, or for three‐dimensional electron tomography. Cells were cultured on laser‐sintered Ti‐6Al‐4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell‐material cross‐sectional interface at the single cell level across multiple high‐resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell‐material interface.     

Synchrotron tomographic images from human lung adenocarcinoma: Three‐dimensional reconstruction and histologic correlations

High‐resolution tomographic images using synchrotron X‐rays are expected to provide detailed reflection of microstructures, thereby allowing for the examination of histologic structures without destruction of the specimen. This study aims to evaluate the synchrotron tomographic images of mixed ground‐glass opacity excised on 5‐mm sections in comparison to pathologic examination. The Institutional Review Board of our institute approved this retrospective study, and written informed consent was obtained from each patient whose lung tissue would be used. Obtained lung cancer specimens were brought to the multiple Wiggler 6C beam line at the Pohang Light Source (PLS‐II) in Korea, and phase contrast X‐ray images were obtained in November 2016. The X‐ray emanated from a bending magnet of the electron storage ring with electron energy of 3 GeV, and a typical beam current was 320 mA. Reconstructed tomographic images were compared with images from histologic slides obtained from the same samples. Pulmonary microstructures including terminal bronchioles, alveolar sacs, and vasculature were identified with phase contrast X‐ray images. Images from normal lung tissue and mixed ground‐glass opacity were clearly distinguishable. Hyperplasia of the interalveolar septum and dysplasia of microstructure were clearly identified. The imaging findings correlated well with hematoxylin‐eosin stained specimens. Tomographic images using synchrotron radiation have the potential for clinical applications. With refinement, this technique may become a diagnostic tool for detection of lung cancer.     

Three‐dimensional bright‐field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright‐field scanning transmission electron microscopy (BF‐STEM) and in the present report BF‐STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon‐degrading soil bacterium, Delftia acidovorans Cs1‐4. Dual‐axis BF‐STEM enabled high‐resolution 3‐D tomographic recontructions (6–10 nm) visualization of thick (1250 and 1500 nm) sections. The 3‐D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF‐STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual‐axis BF‐STEM tomography to obtain high‐resolution 3‐D images of novel nanostructures in bacterial biofilms. Future work with dual‐axis BF‐STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.     

High-resolution three-dimensional imaging of the rich membrane structures of bone marrow-derived mast cells

Atomic force microscopy (AFM) enables high-resolution three-dimensional (3D) imaging of cultured bone marrow-derived mast cells. Cells were immobilized by a quick centrifugation and fixation to preserve their transient cellular morphologies followed by AFM characterization in buffer. This "fix-and-look" approach preserves the structural integrity of individual cells. Well-known membrane morphologies, such as ridges and microvilli, are visualized, consistent with prior electron microscopy observations. Additional information including the 3D measurements of these characteristic features are attained from AFM topographs. Filopodia and lamellopodia, associated with cell spreading, were captured and visualized in three dimensions. New morphologies are also revealed, such as high-density ridges and micro-craters. This investigation demonstrates that the "fix-and-look" approach followed by AFM imaging provides an effective means to characterize the membrane structure of hydrated cells with high resolution. The quantitative imaging and measurements pave the way for systematic correlation of membrane structural features with the biological status of individual cells.