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.     

Microscopic evidences that bone marrow mononuclear cell treatment improves sciatic nerve regeneration after neurorrhaphy

Cell therapy constitutes a possibility for improving nerve regeneration, increasing the success of nerve repair. We evaluate the use of mononuclear cells in the regeneration of the sciatic nerve after axotomy followed by end‐to‐end neurorrhaphy. Forty adult male Wistar rats (250–300 g) were divided into four groups: (1) sham, (2) neurorrhaphy: the sciatic nerve was sectioned and repaired using epineural sutures, (3) culture medium: after the suture, received an injection of 10 μL of culture medium into the nerve, and (4) mononuclear cell: after the suture, a concentration of 3 × 10⁶ of mononuclear cell was injected in epineurium region. Mononuclear cells were obtained from the bone marrow aspirates and separated by Ficoll‐Hypaque method. The histological analyses were performed at the 4th postoperative day. The sciatic functional index, histological, and morphometric analyzes were used to evaluate nerve regeneration at the 6th postoperative week. Six rats were used for immunohistochemical analysis on the 4th postoperative day. In the group 4, on the fourth day, the histological analysis demonstrated a more accelerated degenerative process and an increase of the neurotrophic factors was observed. In the 6th week, all the morphometric results of the group 4 were statistically better compared with groups 2 and 3. There was a statistically significant improvement in the sciatic functional index for group 4 compared with groups 2 and 3. Mononuclear cells stimulated nerve regeneration, most probably by speeding up the Wallerian degeneration process as well as stimulating the synthesis of neurotrophic factors. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

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.     

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.     

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.     

Imaging biofilm in porous media using X-ray computed microtomography

In this study, a new technique for three-dimensional imaging of biofilm within porous media using X-ray computed microtomography is presented. Due to the similarity in X-ray absorption coefficients for the porous media (plastic), biofilm and aqueous phase, an X-ray contrast agent is required to image biofilm within the experimental matrix using X-ray computed tomography. The presented technique utilizes a medical suspension of barium sulphate to differentiate between the aqueous phase and the biofilm. Potassium iodide is added to the suspension to aid in delineation between the biofilm and the experimental porous medium. The iodide readily diffuses into the biofilm while the barium sulphate suspension remains in the aqueous phase. This allows for effective differentiation of the three phases within the experimental systems utilized in this study. The behaviour of the two contrast agents, in particular of the barium sulphate, is addressed by comparing two-dimensional images of biofilm within a pore network obtained by (1) optical visualization and (2) X-ray absorption radiography. We show that the contrast mixture provides contrast between the biofilm, the aqueous-phase and the solid-phase (beads). The imaging method is then applied to two three-dimensional packed-bead columns within which biofilm was grown. Examples of reconstructed images are provided to illustrate the effectiveness of the method. Limitations and applications of the technique are discussed. A key benefit, associated with the presented method, is that it captures a substantial amount of information regarding the topology of the pore-scale transport processes. For example, the quantification of changes in porous media effective parameters, such as dispersion or permeability, induced by biofilm growth, is possible using specific upscaling techniques and numerical analysis. We emphasize that the results presented here serve as a first test of this novel approach; issues with accurate segmentation of the images, optimal concentrations of contrast agents and the potential need for use of synchrotron radiation sources need to be addressed before the method can be used for precise quantitative analysis of biofilm geometry in porous media.     

Assessment of In vivo behavior of polymer tube nerve grafts simultaneously with the peripheral nerve regeneration process using scanning electron microscopy technique

In this study, scanning electron microscopy (SEM) has been applied for instantaneous assessment of processes occurring at the site of regenerating nerve. The technique proved to be especially useful when an artificial implant should have been observed but have not yet been extensively investigated before for assessment of nerve tissue. For in vivo studies, evaluation of implant's morphology and its neuroregenerative properties is of great importance when new prototype is developed. However, the usually applied histological techniques require separate and differently prepared samples, and therefore, the results are never a 100% comparable. In our research, we found SEM as a technique providing detailed data both on an implant behavior and the nerve regeneration process inside the implant. Observations were carried out during 12‐week period on rat sciatic nerve injury model reconstructed with nerve autografts and different tube nerve grafts. Samples were analyzed with haematoxylin‐eosin (HE), immunocytochemical staining for neurofillament and S‐100 protein, SEM, TEM, and the results were compared. SEM studies enabled to obtain characteristic pictures of the regeneration process similarly to TEM and histological studies. Schwann cell transformation and communication as well as axonal outgrowth were identified, newly created and matured axons could be recognized. Concurrent analysis of biomaterial changes in the implant (degradation, collapsing of the tube wall, migration of alginate gel) was possible. This study provides the groundwork for further use of the described technique in the nerve regeneration studies. SCANNING 35: 232‐245, 2013. © 2012 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.     

Microglia in neuroregeneration

Microglia has the potential to produce and release a range of factors that directly and/or indirectly promote regeneration in the injured nervous system. The overwhelming evidence indicates, however, that this potential is generally not expressed in vivo. Activated microglia may enhance neuronal degeneration following axotomy, thereby counteracting functional recovery. Microglia does not seem to contribute significantly to axonal outgrowth after peripheral nerve injury, since this process proceeds uneventful even if perineuronal microglia is eliminated. The phagocytic phenotype of microglia is highly suppressed during Wallerian degeneration in the central nervous system. Therefore, microglia is incapable of rapid and efficient removal of myelin debris and its putative growth inhibitory components. In this way, microglia may contribute to regeneration failure in the central nervous system. Structural and temporal correlations are compatible with participation by perineuronal microglia in axotomy-induced shedding of presynaptic terminals, but direct evidence for such participation is lacking. Currently, the most promising case for a promoting effect on neural repair by activated microglia appears to be as a mediator of collateral sprouting, at least in certain brain areas. However, final proof for a critical role of microglia in these instances is still lacking. Results from in vitro studies demonstrate that microglia can develop a regeneration supportive phenotype. Altering the microglial involvement following neural injury from a typically passive or even counterproductive state and into a condition where these cells are actively supporting regeneration and plasticity is, therefore, an exciting challenge and probably a realistic goal.     

Strain mapping accuracy improvement using super‐resolution techniques

Super‐resolution (SR) software‐based techniques aim at generating a final image by combining several noisy frames with lower resolution from the same scene. A comparative study on high‐resolution high‐angle annular dark field images of InAs/GaAs QDs has been carried out in order to evaluate the performance of the SR technique. The obtained SR images present enhanced resolution and higher signal‐to‐noise (SNR) ratio and sharpness regarding the experimental images. In addition, SR is also applied in the field of strain analysis using digital image processing applications such as geometrical phase analysis and peak pairs analysis. The precision of the strain mappings can be improved when SR methodologies are applied to experimental images.     

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.     

A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation

Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.     

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.     

Improved Three-Dimensional Image Reconstruction Technique for Multi-Component Ground Penetrating Radar Data

For electromagnetic imaging the vectorial character of the emitted field and the radiation characteristics of both source and receiver play an important role. Recently, a new imaging algorithm was presented dedicated to the electromagnetic case. The radiation characteristics of GPR source and receiver antennas and the vectorial nature of the electromagnetic waves are taken into account for a monostatic fixed offset GPR survey. This resulted in a representative image of a point scatterer. Comparison with scalar imaging algorithms shows that for a homogeneous space the SAR image has an opposite sign compared to the multi-component image, whereas the Gazdag image has a phase shift of about 90° with respect to the multi-component image. In this paper, modified scalar imaging algorithms are introduced that minimize these differences. However, between the images obtained in a homogeneous half-space phase differences of 10–20° are still present. These differences indicate the possible error in nature of the physical property contrast when it is determined with the modified scalar imaging algorithms. The multi-component imaging algorithm returns a representative image because it uses the appropriate Greens functions to eliminate the propagation effects. For practical imaging strategies, only far-field radiation characteristics can be used to compensate for the propagation effects due to the large computing time needed to evaluate the total-field expressions. Synthetic analysis of the imaging of a point scatterer calculated using total-field expressions shows that using the far-field expressions in the multi-component imaging algorithm the images better approximate the actual contrast than using the modified scalar imaging algorithms. Experimental results are presented from imaging several buried objects with different medium properties and different orientations. The phase differences in the experimental data are similar to those obtained synthetically. This likeness indicates that using the multi-component imaging algorithm, a more reliable image is obtained than with the modified scalar imaging algorithms.     

Histological and electrophysiological analysis of the peripheral nerve allografts using an immunosuppressive agent

In peripheral nerve allografts, use of an immunosuppressive agent is one of the ways of reducing nerve rejection. FK506 is a newly discovered substance, extracted from Streptomyces tsukubaensis, and has strong immunosuppressive effects. In the present study, immunosuppressive effects of FK 506 were examined using allografts of rat sciatic nerves. Good nerve regeneration was demonstrated through 12 weeks in this model. The immunosuppressed group gained weight over the course of the experiment. Another study was performed to observe the histological changes caused by ceasing the administration of FK506. Administration of FK506 was terminated 12 weeks after grafting. At 8 weeks after cessation, cellular infiltration and large unmyelinated axons were observed in the extended subperineurial space of grafts. At 12 weeks, histological characteristics of rejection were not observed. In the electrophysiological study, the temporal dispersions were recorded at 4 and 8 weeks. However, the normal electrophysiological waves were recorded at 12 weeks after cessation. It was concluded that FK506 is effective for preventing rejection of nerve allografts without any serious side effects on rats, and findings of total rejection of grafts were not recognized after ceasing the administration of FK 506. In peripheral nerve allografts, short-term administration of an immunosuppressive agent is sufficient to lead to good nerve regeneration.     

Phase and amplitude contrast in electron microscopy of stained biological objects.

For biological objects negatively stained with heavy atom material, electron microscope images show best contrast for image detail on the scale of 10--20 A when a small objective aperture is used. In images taken under the optimum phase contrast imaging conditions of Scherzer, the required image detail is lost in unwanted noise. Both of these conditions may be described in terms of phase contrast imaging for a thin phase object. Calculations of image intensities and noise are reported for a model object consisting of heavy and light atoms randomly distributed to simulate a negatively stained protein molecule. The results are consistent with experimental observations.     

Minima equalization: A useful strategy in automatic processing of microscopic images

Specifically stained features in microscopic images rarely have a unique range of grey levels which would permit selection of the features by simple thresholding. The spaces between features and immediately surrounding them are often as dark or darker than the more lightly stained features. An algorithm for minima equalization which facilitates the extraction and segmentation of such features was designed and is explained in intuitive terms. It is applied to the analysis of cross-sections of peripheral myelinated nerve fibres. It is shown that the binary image obtained can be combined with a gradient image to give a binary image which accurately reflects the thickness of the myelin in the original image. Using silver-impregnated nerve endings and bile canaliculi stained for thiamine pyrophosphatase, binary images like those prepared manually from thick specimens using a camera lucida can be obtained using the minima equalization procedure. The image processor is used to develop a composite image by combining images at various focal planes through the thick specimen. This image is then processed to obtain the binary image.     

Improved Zernike‐type phase contrast for transmission electron microscopy

Zernike phase contrast has been recognized as a means of recording high‐resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image typical phase objects such as unstained biological molecules or cryosections of biological tissue. According to the original proposal discussed in Danev and Nagayama (2001) and references therein, the Zernike phase plate applies a phase shift of π/2 to all scattered electron beams outside a given scattering angle and an image is recorded at Gaussian focus or slight underfocus (below Scherzer defocus). Alternatively, a phase shift of ‐π/2 is applied to the central beam using the Boersch phase plate. The resulting image will have an almost perfect contrast transfer function (close to 1) from a given lowest spatial frequency up to a maximum resolution determined by the wave length, the amount of defocus and the spherical aberration of the microscope. In this paper, I present theory and simulations showing that this maximum spatial frequency can be increased considerably without loss of contrast by using a Zernike or Boersch phase plate that leads to a phase shift between scattered and unscattered electrons of only π /4, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies.