Micro‐CT versus synchrotron radiation phase contrast imaging of human cochlea

High‐resolution images of the cochlea are used to develop atlases to extract anatomical features from low‐resolution clinical computed tomography (CT) images. We compare visualization and contrast of conventional absorption‐based micro‐CT to synchrotron radiation phase contrast imaging (SR‐PCI) images of whole unstained, nondecalcified human cochleae. Three cadaveric cochleae were imaged using SR‐PCI and micro‐CT. Images were visually compared and contrast‐to‐noise ratios (CNRs) were computed from n = 27 regions‐of‐interest (enclosing soft tissue) for quantitative comparisons. Three‐dimensional (3D) models of cochlear internal structures were constructed from SR‐PCI images using a semiautomatic segmentation method. SR‐PCI images provided superior visualization of soft tissue microstructures over conventional micro‐CT images. CNR improved from 7.5 ± 2.5 in micro‐CT images to 18.0 ± 4.3 in SR‐PCI images (p < 0.0001). The semiautomatic segmentations yielded accurate reconstructions of 3D models of the intracochlear anatomy. The improved visualization, contrast and modelling achieved using SR‐PCI images are very promising for developing atlas‐based segmentation methods for postoperative evaluation of cochlear implant surgery.     

铝质滤波片在微聚焦钨靶X射线源相衬成像中的应用研究

实验室建立了以钨靶为光源的微聚焦X射线相衬成像系统,为减少应用过程中微聚焦光源的多色性对系统成像质量的影响,使用一系列不同厚度的铝质滤波片对弱吸收材料聚丙烯吸管进行微聚焦X射线相衬成像的研究。结果说明铝质滤波片能够改善成像质量,且随着铝质滤波片厚度的增加图像中物体的边缘特征的衬度呈指数衰减。为铝质滤波片在钨靶微聚焦X射线相衬成像系统中的应用提供了经验。     

Soft X-ray contact imaging of nucleolar chromatin using synchrotron radiation: a comparative scanning and transmission electron microscope study

Contact images (CI) of dehydrated, nucleolar chromatin from amphibian oocytes have been produced by soft X-rays from a synchrotron radiation source. These CI have been compared with the morphology of the original chromatin as seen in scanning and transmission electron microscopes. The quality and informational content of the CI depend very much on certain preparative procedures. The following factors have a marked effect on image quality and need to be carefully controlled: the total X-ray dose, the time and nature of development and the distance of the specimen from the photoresist. The preparation of the chromatin itself, providing that it is critically point dried, is less important. By following a regime of high X-ray dose, sufficient for penetration of the rather thick chromatin rings, and gentle development so that fine detail is not dissolved from the resist surface, it has been possible to obtain images which closely resemble the original chromatin, although the detailed resolution of the CI is not as clear. The smallest biological structures clearly resolved in the CI are ribonucleoprotein granules, which vary in size from 200 to 800 nm. However, by further refinement of preparative conditions it should be possible to improve on the informational content of these images.     

High-resolution X-ray refraction imaging of rat lung and histological correlations

This study was performed to observe microstructures of the rat lung, using a synchrotron radiation beam and to compare findings with histological observations. X-ray refraction images from ex-vivo ventilating rat lung were obtained with an 8 KeV monochromatic beam and 20-mum thick CsI(Tl) scintillation crystal. The visual image was magnified using a 20x microscope objective and captured using an analog CCD camera. Obtained images were compared with conventional light microscopic findings from the same tissue. Pulmonary microstructures, including alveolar ducts, alveolar sacs, alveoli, alveolar walls, and perialveolar capillary networks were clearly identified with spatial resolution of as much as 1.2 mum and had good correlation with conventional light microscopic findings. The shape of alveoli appeared more round in SR images than in the light microscopic images. The results suggest that X-ray microscopy study of the lung using synchrotron radiation demonstrates the potential for clinically relevant microstructure of lung tissue without sectioning and fixation.     

Phase contrast without phase plates and phase rings—optical solutions for improved imaging of phase structures

Using the optical methods described, phase specimens can be observed with a modified light microscope in enhanced clarity, purified from typical artifacts which are apparent in standard phase contrast illumination. In particular, haloing and shade‐off are absent, lateral and vertical resolution are maximized and the image quality remains constant even in problematic preparations which cannot be well examined in normal phase contrast, such as specimens beyond a critical thickness or covered by obliquely situated cover slips. The background brightness and thus the range of contrast can be continuously modulated and specimens can be illuminated in concentric‐peripheral, axial or paraxial light. Additional contrast effects can be achieved by spectral color separation. Normal glass or mirror lenses can be used; they do not need to be fitted with a phase plate or a phase ring. The methods described should be of general interest for all disciplines using phase microscopy. Microsc. Res. Tech., 76:1050–1056, 2013. © 2013 Wiley Periodicals, Inc.     

Regularized phase tomography enables study of mineralized and unmineralized tissue in porous bone scaffold

Regularized phase tomography was used to image non‐calcified fibrous matrix in in vitro cell‐cultivated porous bone scaffold samples. 3D micro‐architecture of bone and bone scaffold has previously been studied by micro‐computed tomography, synchrotron radiation (SR) micro‐computed tomography and microdiffraction. However, neither of these techniques can resolve the low‐calcified immature pre‐bone fibrous structures. Skelite porous scaffold discs were seeded with osteoblasts, a combination of osteoblast and pre‐osteoclasts and, as controls, with pre‐osteoclasts only, and then cultivated for 8 weeks. They were subsequently imaged using SR propagation‐based phase contrast imaging. Reconstructions using a regularized holographic phase tomography approach were compared to standard (absorption) SR micro‐computed tomography, which show that quantitative analysis, such as volume and thickness measurements, of both the calcified fraction and the immature bone matrix in the reconstructed volumes is enabled. Indications of the effect of this type of culture on Skelite, such as change in mineralization and deposit of mature bone on the walls of the scaffold, are found. The results are verified with a histological study.     

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.     

Confocal differential interference contrast (DIC) microscopy: including a theoretical analysis of conventional and confocal DIC imaging

A technique for obtaining differential interference contrast (DIC) imaging using a confocal microscope system is examined and its features compared to those of existing confocal differential phase contrast (DPC) techniques as well as to conventional Nomarski DIC. A theoretical treatment of DIC imaging is presented, which takes into account the vignetting effect caused by the finite size of the lens pupils. This facilitates the making of quantitative measurements in DIC and allows the user to identify and select the most appropriate system parameters, such as the bias retardation and lateral shear of the Wollaston prism.     

Application of sensitive,high‐resolution imaging at a commercial lab‐based X‐ray micro‐CT system using propagation‐based phase retrieval

Several dedicated commercial lab‐based micro‐computed tomography (μCT) systems exist, which provide high‐resolution images of samples, with the capability to also deliver in‐line phase contrast. X‐ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single‐distance phase‐contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high‐quality images, measured without the use of a synchrotron X‐ray source, demonstrate that highly sensitive, micrometre‐resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.     

A new sample preparation method for biological soft X-ray microscopy: nitrogen-based contrast and radiation tolerance properties of glycol methacrylate-embedded and sectioned tissue

We describe the preparation of a biological tissue for imaging in a transmission soft X-ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual-wavelength imaging at the nitrogen K-shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large-scale X-ray microscopic study on sections of plastic-embedded soft tissue. At radiation doses of up to 10⁸ Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X-ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample.