X-ray microscopy of plant cells by using LiF crystal as a detector

A lithium fluoride (LiF) crystal has been utilized as a new soft X-ray detector to image different biological samples at a high spatial resolution. This new type of image detector for X-ray microscopy has many interesting properties: high resolution (nanometer scale), permanent storage of images, the ability to clear the image and reuse the LiF crystal, and high contrast with greater dynamic range. Cells of the unicellular green algae Chlamydomonas dysosmos and Chlorella sorokiniana, and pollen grains of Olea europea have been used as biological materials for imaging. The biological samples were imaged on LiF crystals by using the soft X-ray contact microscopy and contact micro-radiography techniques. The laser plasma soft X-ray source was generated using a Nd:YAG/Glass laser focused on a solid target. The X-ray energy range for image acquisition was in the water-window spectral range for single shot contact microscopy of very thin biological samples (single cells) and around 1 keV for multishots microradiography. The main aim of this article is to highlight the possibility of using a LiF crystal as a detector for the biological imaging using soft X-ray radiation and to demonstrate its ability to visualize the microstructure within living cells.     

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.     

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.     

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.     

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.     

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.     

Exploring the use of soft X‐ray microscopy for imaging subcellular structures of the inner ear1

The soft X‐ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X‐ray microscopy provides high‐resolution visualization of hydrated, non‐embedded and non‐sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X‐ray micrographs of structures isolated from the guinea‐pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X‐ray images of the acellular tectorial membrane and thin two‐layered Reissner's membrane display a level of resolution comparable to low‐power electron microscopy.     

Comparison of single‐particle analysis and electron tomography approaches: an overview

Three‐dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X‐ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three‐dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single‐particle analysis. The first one is currently used to elucidate the three‐dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.     

High-resolution compact X-ray microscopy

We demonstrate compact full‐field soft X‐ray transmission microscopy with sub 60‐nm resolution operating at λ= 2.48 nm. The microscope is based on a 100‐Hz regenerative liquid‐nitrogen‐jet laser‐plasma source in combination with a condenser zone plate and a micro‐zone plate objective for high‐resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre‐aligned using a visible‐light microscope. High‐quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples.     

Evaluation of contrasting techniques for X‐ray imaging of velvet worms (Onychophora)

Non‐invasive imaging techniques like X‐ray computed tomography have become very popular in zoology, as they allow for simultaneous imaging of the internal and external morphology of organisms. Nevertheless, the effect of different staining approaches required for this method on samples lacking mineralized tissues, such as soft‐bodied invertebrates, remains understudied. Herein, we used synchrotron radiation‐based X‐ray micro‐computed tomography to compare the effects of commonly used contrasting approaches on onychophorans – soft‐bodied invertebrates important for studying animal evolution. Representatives of Euperipatoides rowelli were stained with osmium tetroxide (vapour or solution), ruthenium red, phosphotungstic acid, or iodine. Unstained specimens were imaged using both standard attenuation‐based and differential phase‐contrast setups to simulate analyses with museum material. Our comparative qualitative analyses of several tissue types demonstrate that osmium tetroxide provides the best overall tissue contrast in onychophorans, whereas the remaining staining agents rather favour the visualisation of specific tissues and/or structures. Quantitative analyses using signal‐to‐noise ratio measurements show that the level of image noise may vary according to the staining agent and scanning medium selected. Furthermore, box‐and‐whisker plots revealed substantial overlap in grey values among structures in all datasets, suggesting that a combination of semiautomatic and manual segmentation of structures is required for comprehensive 3D reconstructions of Onychophora, irrespective of the approach selected. Our results show that X‐ray micro‐computed tomography is a promising technique for studying onychophorans and, despite the benefits and disadvantages of different staining agents for specific tissues/structures, this method retrieves informative data that may eventually help address evolutionary questions long associated with Onychophora.     

X-ray high-resolution vascular network imaging

This paper presents the first application of high‐resolution X‐ray synchrotron tomography to the imaging of large microvascular networks in biological tissue samples. This technique offers the opportunity of analysing the full three‐dimensional vascular network from the micrometre to the millimetre scale. This paper presents the specific sample preparation method and the X‐ray imaging procedure. Either barium or iron was injected as contrast agent in the vascular network. The impact of the composition and concentration of the injected solution on the X‐ray synchrotron tomography images has been studied. Two imaging modes, attenuation and phase contrast, are compared. Synchrotron high‐resolution computed tomography offers new prospects in the three‐dimensional imaging of in situ biological vascular networks.     

Quantitative X-ray projection microscopy: phase-contrast and multi-spectral imaging

We outline a new approach to X‐ray projection microscopy in a scanning electron microscope (SEM), which exploits phase contrast to boost the quality and information content of images. These developments have been made possible by the combination of a high‐brightness field‐emission gun (FEG)‐based SEM, direct detection CCD technology and new phase retrieval algorithms. Using this approach we have been able to obtain spatial resolution of < 0.2 µm and have demonstrated novel features such as: (i) phase‐contrast enhanced visibility of high spatial frequency image features (e.g. edges and boundaries) over a wide energy range; (ii) energy‐resolved imaging to simultaneously produce multiple quasi‐monochromatic images using broad‐band polychromatic illumination; (iii) easy implementation of microtomography; (iv) rapid and robust phase/amplitude‐retrieval algorithms to enable new real‐time and quantitative modes of microscopic imaging. These algorithms can also be applied successfully to recover object–plane information from intermediate‐field images, unlocking the potentially greater contrast and resolution of the intermediate‐field regime. Widespread applications are envisaged for fields such as materials science, biological and biomedical research and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to projection microscopy using other sources of radiation, such as visible light and electrons.     

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.     

Visualization of water drying in porous materials by X‐ray phase contrast imaging

We present in this study results from X‐ray tomographic microscopy with synchrotron radiation performed both in attenuation and phase contrast modes on a limestone sample during two stages of water drying. No contrast agent was used in order to increase the X‐ray attenuation by water. We show that only by using the phase contrast mode it is possible to achieve enough water content change resolution to investigate the drying process at the pore‐scale. We performed 3D image analysis of the time‐differential phase contrast tomogram. We show by the results of such analysis that it is possible to obtain a reliable characterization of the spatial redistribution of water in the resolved pore system in agreement with what expected from the theory of drying in porous media and from measurements performed with other approaches. We thus show the potential of X‐ray phase contrast imaging for pore‐scale investigations of reactive water transport processes which cannot be imaged by adding a contrast agent for exploiting the standard attenuation contrast imaging mode.     

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.     

Soft X‐ray spectromicroscopy for speciation,quantitation and nano‐eco‐toxicology of nanomaterials

There is a critical need for methods that provide simultaneous detection, identification, quantitation and visualization of nanomaterials at their interface with biological and environmental systems. The approach should allow speciation as well as elemental analysis. Using the intrinsic X‐ray absorption properties, soft X‐ray scanning transmission X‐ray spectromicroscopy (STXM) allows characterization and imaging of a broad range of nanomaterials, including metals, oxides and organic materials, and at the same time is able to provide detailed mapping of biological components. Thus, STXM offers considerable potential for application to research on nanomaterials in biology and the environment. The potential and limitations of STXM in this context are discussed using a range of examples, focusing on the interaction of nanomaterials with microbial cells, biofilms and extracellular polymers. The studies outlined include speciation and mapping of metal‐containing nanomaterials (Ti, Ni, Cu) and carbon‐based nanomaterials (multiwalled carbon nanotubes, C₆₀ fullerene). The benefits of X‐ray fluorescence detection in soft X‐ray STXM are illustrated with a study of low levels of Ni in a natural river biofilm.     

Atomic force microscopy imaging of actin cortical cytoskeleton of Xenopus laevis oocyte

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside‐out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X‐100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high‐resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high‐resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins.     

Iodine vapor staining for atomic number contrast in backscattered electron and X‐ray imaging

Iodine imparts strong contrast to objects imaged with electrons and X‐rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation‐deposition state‐change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas‐tight container along with a small mass of solid I₂. The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin‐embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X‐ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron‐ and photon‐sample interactions, such as transmission electron microscopy and light microscopy. Microsc. Res. Tech. 77:1044–1051, 2014. © 2014 The Authors. Microscopy Research Technique published by Wiley Periodocals, Inc.