Imaging of Mouse Brain Fixated in Ethanol in Micro-CT.

Micro-CT imaging is a well-established morphological method for the visualization of animal models. We used ethanol fixation of the mouse brains to perform high-resolution micro-CT scans showing in great details brain grey and white matters. It was possible to identify more than 50 neuroanatomical structures on the 5 selected coronal sections. Among white matter structures, we identified fornix, medial lemniscus, crossed tectospinal pathway, mammillothalamic tract, and the sensory root of the trigeminal ganglion. Among grey matter structures, we identified basal nuclei, habenular complex, thalamic nuclei, amygdala, subparts of hippocampal formation, superior colliculi, Edinger-Westphal nucleus, and others. We suggest that micro-CT of the mouse brain could be used for neurohistological lesions evaluation as an alternative to classical neurohistology because it does not destroy brain tissue.

Ethanol fixation method for heart and lung imaging in micro-CT.

PURPOSE: The soft tissue imaging in micro-CT remains challenging due to its low intrinsic contrast. The aim of this study was to create a simple staining method omitting the usage of contrast agents for ex vivo soft tissue imaging in micro-CT. MATERIALS AND METHODS: Hearts and lungs from 30 mice were used. Twenty-seven organs were either fixed in 97% or 50% ethanol solution or in a series of ascending ethanol concentrations. Images were acquired after 72, 168 and 336 h on a custom-built micro-CT machine and compared to scans of three native samples. RESULTS: Ethanol provided contrast enhancement in all evaluated fixations. Fixation in 97% ethanol resulted in contrast enhancement after 72 h; however, it caused hardening of the samples. Fixation in 50% ethanol provided contrast enhancement after 336 h, with milder hardening, compared to the 97% ethanol fixation, but the visualization of details was worse. The fixation in a series of ascending ethanol concentrations provided the most satisfactory results; all organs were visualized in great detail without tissue damage. CONCLUSIONS: Simple ethanol fixation improves the tissue contrast enhancement in micro-CT. The best results can be obtained with fixation of the soft tissue samples in a series of ascending ethanol concentrations.

Non-destructive imaging of fragments of historical beeswax seals using high-contrast X-ray micro-radiography and micro-tomography with large area photon-counting detector array.

Historical beeswax seals are unique cultural heritage objects. Unfortunately, a number of historical sealing waxes show a porous structure with a strong tendency to stratification and embrittlement, which makes these objects extremely prone to mechanical damage. The understanding of beeswax degradation processes therefore plays an important role in the preservation and consequent treatment of these objects. Conventional methods applied for the investigation of beeswax materials (e.g. gas chromatography) are of a destructive nature or bring only limited information about the sample surface (microscopic techniques). Considering practical limitations of conventional methods and ethical difficulties connected with the sampling of the historical material, radiation imaging methods such as X-ray micro-tomography presents a promising non-destructive tool for the onward scientific research in this field. In this contribution, we present the application of high-contrast X-ray micro-radiography and micro-tomography for the investigation of beeswax seal fragments. The method is based on the application of the large area photon-counting detector recently developed at our institute. The detector combines the advantages of single-photon counting technology with a large field of view. The method, consequently, enables imaging of relatively large objects with high geometrical magnification. In the reconstructed micro-tomographies of investigated historical beeswax seals, we are able to reveal morphological structures such as stratification, micro-cavities and micro-fractures with spatial resolution down to 5µm non-destructively and with high imaging quality. The presented work therefore demonstrates that a combination of state-of-the-art hybrid pixel semiconductor detectors and currently available micro-focus x-ray sources makes it possible to apply X-ray micro-radiography and micro-tomography as a valuable non-destructive tool for volumetric beeswax seal morphological studies.

High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector.

Using dedicated contrast agents high-quality X-ray imaging of soft tissue structures with isotropic micrometre resolution has become feasible. This technique is frequently titled as virtual histology as it allows production of slices of tissue without destroying the sample. The use of contrast agents is, however, often an irreversible time-consuming procedure and despite the non-destructive principle of X-ray imaging, the sample is usually no longer usable for other research methods. In this work we present the application of recently developed large-area photon counting detector for high resolution X-ray micro-radiography and micro-tomography of whole ex-vivo ethanol-preserved mouse organs. The photon counting detectors provide dark-current-free quantum-counting operation enabling acquisition of data with virtually unlimited contrast-to-noise ratio (CNR). Thanks to the very high CNR even ethanol-only preserved soft-tissue samples without addition of any contrast agent can be visualized in great detail. As ethanol preservation is one of the standard steps of tissue fixation for histology, the presented method can open a way for widespread use of micro-CT with all its advantages for routine 3D non-destructive soft-tissue visualisation.

Trichobilharzia regenti (Schistosomatidae): 3D imaging techniques in characterization of larval migration through the CNS of vertebrates.

Migration of parasitic worms through the host tissues, which may occasionally result in fatal damage to the internal organs, represents one of the major risks associated with helminthoses. In order to track the parasites, traditionally used 2D imaging techniques such as histology or squash preparation do not always provide sufficient data to describe worm location/behavior in the host. On the other hand, 3D imaging methods are widely used in cell biology, medical radiology, osteology or cancer research, but their use in parasitological research is currently occasional. Thus, we aimed at the evaluation of suitability of selected 3D methods to monitor migration of the neuropathogenic avian schistosome Trichobilharzia regenti in extracted spinal cord of experimental vertebrate hosts. All investigated methods, two of them based on tracking of fluorescently stained larvae with or without previous chemical clearing of tissue and one based on X-ray micro-CT, exhibit certain limits for in vivo observation. Nevertheless, our study shows that the tested methods as ultramicroscopy (used for the first time in parasitology) and micro-CT represent promising tool for precise analyzing of parasite larvae in the CNS. Synthesis of these 3D imaging techniques can provide more comprehensive look at the course of infection, host immune response and pathology caused by migrating parasites within entire tissue samples, which would not be possible with traditional approaches.

Hard x-ray phase contrast imaging using single absorption grating and hybrid semiconductor pixel detector.

A method for x-ray phase contrast imaging is introduced in which only one absorption grating and a microfocus x-ray source in a tabletop setup are used. The method is based on precise subpixel position determination of the x-ray pattern projected by the grating directly from the pattern image. For retrieval of the phase gradient and absorption image (both images obtained from one exposure), it is necessary to measure only one projection of the investigated object. Thus, our method is greatly simplified compared with the phase-stepping method and our method can significantly reduce the time-consuming scanning and possibly the unnecessary dose. Furthermore, the technique works with a fully polychromatic spectrum and gives ample variability in object magnification. Consequently, the approach can open the way to further widespread application of phase contrast imaging, e.g., into clinical practice. The experimental results on a simple testing object as well as on complex biological samples are presented.