Peter Thalmann

Single and double grating-based X-ray microtomography using synchrotron radiation

Hard X-ray phase contrast imaging techniques have become most suitable for the non-destructive three-dimensional visualization of soft tissues at the microscopic level. Among the hard X-ray grating interferometry methods, a single-grating approach (XSGI) has been implemented by simplifying the established double-grating interferometer (XDGI). We quantitatively compare the XSGI and XDGI tomograms of a human nerve and demonstrate that both techniques provide sufficient contrast to allow for the distinction of tissue types. The two-fold binned data show spatial resolution of (5.2 ± 0.6) μm and (10.7 ± 0.6) μm, respectively, underlying the performance of XSGI in soft tissue imaging.

Mineralization of Early Stage Carious Lesions In Vitro—A Quantitative Approach

Micro computed tomography has been combined with dedicated data analysis for the in vitro quantification of sub-surface enamel lesion mineralization. Two artificial white spot lesions, generated on a human molar crown in vitro, were examined. One lesion was treated with a self-assembling peptide intended to trigger nucleation of hydroxyapatite crystals. We non-destructively determined the local X-ray attenuation within the specimens before and after treatment. The three-dimensional data was rigidly registered. Three interpolation methods, i.e., nearest neighbor, tri-linear, and tri-cubic interpolation were evaluated. The mineralization of the affected regions was quantified via joint histogram analysis, i.e., a voxel-by-voxel comparison of the tomography data before and after mineralization. After ten days incubation, the mean mineralization coefficient reached 35.5% for the peptide-treated specimen compared to 11.5% for the control. This pilot study does not give any evidence for the efficacy of peptide treatment nor allows estimating the necessary number of specimens to achieve significance, but shows a sound methodological approach on the basis of the joint histogram analysis.

Three-dimensional and non-destructive characterization of nerves inside conduits using laboratory-based micro computed tomography

BACKGROUND Histological assessment of peripheral nerve regeneration in animals is tedious, time-consuming and challenging for three-dimensional analysis. NEW METHOD The present study reports on how and to what extent micro computed tomography of paraffin-embedded samples can provide a reliable three-dimensional approach for quantitative analysis of peripheral nerves. RESULTS Rat sciatic nerves were harvested, formalin-fixated, positioned into nerve conduits (NC), paraffin-embedded, and imaged using a laboratory-based X-ray microtomography system with an isotropic voxel length of 4μm. Suitable quantitative measures were identified and automatically evaluated, i.e. nerve length, cross-sectional area and volume, as well as vascular structures, to be used as an assessment and comparison indicator of regeneration quality. COMPARISON WITH EXISTING METHODS Compared to imaging using contrast agents, the investigated specimens can subsequently undergo the conventional histological analysis without requiring additional preparation steps. Contrast and spatial resolution are also increased significantly. CONCLUSIONS We demonstrate the potential of the micro computed tomography for non-destructive monitoring of peripheral nerves inside the conduits.

Implementation of a double-grating interferometer for phase-contrast computed tomography in a conventional system nanotom® m

Visualizing the internal architecture of large soft tissue specimens within the laboratory environment in a label-free manner is challenging, as the conventional absorption-contrast tomography yields a poor contrast. In this communication, we present the integration of an X-ray double-grating interferometer (XDGI) into an advanced, commercially available micro computed tomography system nanotom® m with a transmission X-ray source and a micrometer-sized focal spot. The performance of the interferometer is demonstrated by comparing the registered three-dimensional images of a human knee joint sample in phase- and conventional absorption-contrast modes. XDGI provides enough contrast (1.094 ± 0.152) to identify the cartilage layer, which is not recognized in the conventional mode (0.287 ± 0.003). Consequently, the two modes are complementary, as the present XDGI set-up only reaches a spatial resolution of (73 ± 6) μm, whereas the true micrometer resolution in the absorption-contrast mode has been proven. By providing complimentary information, XDGI is especially a supportive quantitative method for imaging soft tissues and visualizing weak X-ray absorbing species in the direct neighborhood of stronger absorbing components at the microscopic level.

Comparing natural and artificial carious lesions in human crowns by means of conventional hard x-ray micro-tomography and two-dimensional x-ray scattering with synchrotron radiation

Dental caries, one of the most prevalent infectious bacterial diseases in the world, is caused by specific types of acid-producing bacteria. Caries is a disease continuum resulting from the earliest loss of ions from apatite crystals through gross cavitation. Enamel dissolution starts when the pH-value drops below 5.5. Neutralizing the pH-value in the oral cavity opposes the process of demineralization, and so caries lesions occur in a dynamic cyclic de-mineralizing/remineralizing environment. Unfortunately, biomimetic regeneration of cavitated enamel is not yet possible, although remineralization of small carious lesions occurs under optimal conditions. Therefore, the development of methods that can regenerate carious lesions, and subsequently recover and retain teeth, is highly desirable. For the present proceedings we analyzed one naturally occurring sub-surface and one artificially produced lesion. For the characterization of artificial and natural lesions micro computed tomography is the method of choice when looking to determine three-dimensional mineral distribution and to quantify the degree of mineralization. In this pilot study we elucidate that the de-mineralized enamel in natural and artificially induced lesions shows comparable X-ray attenuation behavior, thereby implying that the study protocol employed herein seems to be appropriate. Once we know that the lesions are comparable, a series of well-reproducible in vitro experiments on enamel regeneration could be performed. In order to quantify further lesion morphology, the anisotropy of the enamel’s nanostructure can be characterized by using spatially resolved, small-angle X-ray scattering. We wanted to demonstrate that the artificially induced defect fittingly resembles the natural carious lesion.

Hierarchical imaging of the human knee

Among the clinically relevant imaging techniques, computed tomography (CT) reaches the best spatial resolution. Sub-millimeter voxel sizes are regularly obtained. For investigations on true micrometer level lab-based μCT has become gold standard. The aim of the present study is the hierarchical investigation of a human knee post mortem using hard X-ray μCT. After the visualization of the entire knee using a clinical CT with a spatial resolution on the sub-millimeter range, a hierarchical imaging study was performed using a laboratory μCT system nanotom m. Due to the size of the whole knee the pixel length could not be reduced below 65 μm. These first two data sets were directly compared after a rigid registration using a cross-correlation algorithm. The μCT data set allowed an investigation of the trabecular structures of the bones. The further reduction of the pixel length down to 25 μm could be achieved by removing the skin and soft tissues and measuring the tibia and the femur separately. True micrometer resolution could be achieved after extracting cylinders of several millimeters diameters from the two bones. The high resolution scans revealed the mineralized cartilage zone including the tide mark line as well as individual calcified chondrocytes. The visualization of soft tissues including cartilage, was arranged by X-ray grating interferometry (XGI) at ESRF and Diamond Light Source. Whereas the high-energy measurements at ESRF allowed the simultaneous visualization of soft and hard tissues, the low-energy results from Diamond Light Source made individual chondrocytes within the cartilage visual.

Three-dimensional registration of synchrotron radiation-based micro-computed tomography images with advanced laboratory micro-computed tomography data frommurine kidney casts

Malfunction of oxygen regulation in kidney and liver may lead to the pathogenesis of chronic diseases. The underlying mechanisms are poorly understood. In kidney, it is hypothesized that renal gas shunting from arteries to veins eliminates excess oxygen. Such shunting is highly dependent on the structure of the renal vascular network. The vascular tree has so far not been quantified under maintenance of its connectivity as three-dimensional imaging of the vessel tree down to the smallest capillaries, which in mouse model are smaller than 5 μm in diameter, is a challenging task. An established protocol uses corrosion casts and applies synchrotron radiation-based micro-computed tomography (SRμCT), which provides the desired spatial resolution with the necessary contrast. However, SRμCT is expensive and beamtime access is limited. We show here that measurements with a phoenix nanotomrm (General Electric, Wunstorf, Germany) can provide comparable results to those obtained with SRμCT, except for regions with small vessel structures, where the signal-to-noise level was significantly reduced. For this purpose the nanotom®m measurement was compared with its corresponding measurement acquired at the beamline P05 at PETRA III at DESY, Hamburg, Germany.

Grating interferometry-based phase microtomography of atherosclerotic human arteries

Cardiovascular diseases are the number one cause of death and morbidity in the world. Understanding disease development in terms of lumen morphology and tissue composition of constricted arteries is essential to improve treatment and patient outcome. X-ray tomography provides non-destructive three-dimensional data with micrometer-resolution. However, a common problem is simultaneous visualization of soft and hard tissue-containing specimens, such as atherosclerotic human coronary arteries. Unlike absorption based techniques, where X-ray absorption strongly depends on atomic number and tissue density, phase contrast methods such as grating interferometry have significant advantages as the phase shift is only a linear function of the atomic number. We demonstrate that grating interferometry-based phase tomography is a powerful method to three-dimensionally visualize a variety of anatomical features in atherosclerotic human coronary arteries, including plaque, muscle, fat, and connective tissue. Three formalin-fixed, human coronary arteries were measured using advanced laboratory μCT. While this technique gives information about plaque morphology, it is impossible to extract the lumen morphology. Therefore, selected regions were measured using grating based phase tomography, sinograms were treated with a wavelet-Fourier filter to remove ring artifacts, and reconstructed data were processed to allow extraction of vessel lumen morphology. Phase tomography data in combination with conventional laboratory μCT data of the same specimen shows potential, through use of a joint histogram, to identify more tissue types than either technique alone. Such phase tomography data was also rigidly registered to subsequently decalcified arteries that were histologically sectioned, although the quality of registration was insufficient for joint histogram analysis.

Measuring the bending of asymmetric planar EAP structures

The geometric characterization of low-voltage dielectric electro-active polymer (EAP) structures, comprised of nanometer thickness but areas of square centimeters, for applications such as artificial sphincters requires methods with nanometer precision. Direct optical detection is usually restricted to sub-micrometer resolution because of the wavelength of the light applied. Therefore, we propose to take advantage of the cantilever bending system with optical readout revealing a sub-micrometer resolution at the deflection of the free end. It is demonstrated that this approach allows us to detect bending of rather conventional planar asymmetric, dielectric EAP-structures applying voltages well below 10 V. For this purpose, we built 100 μm-thin silicone films between 50 nm-thin silver layers on a 25 μm-thin polyetheretherketone (PEEK) substrate. The increase of the applied voltage in steps of 50 V until 1 kV resulted in a cantilever bending that exhibits only in restricted ranges the expected square dependence. The mean laser beam displacement on the detector corresponded to 6 nm per volt. The apparatus will therefore become a powerful mean to analyze and thereby improve low-voltage dielectric EAP-structures to realize nanometer-thin layers for stack actuators to be incorporated into artificial sphincter systems for treating severe urinary and fecal incontinence.

Three-dimensional imaging and analysis of entire peripheral nerves after repair and reconstruction

BACKGROUND We wanted to achieve a three-dimensional (3D), non-destructive imaging and automatic post-analysis and evaluation of reconstructed peripheral nerves without involving cutting and staining processes. NEW METHOD We used a laboratory-based micro computed tomography system for imaging, as well as a custom analysis protocol. The sample preparation was also adapted in order to achieve 3D images with true micrometer resolution and suitable contrast. RESULTS Analysis of the acquired tomograms enabled the quantitative assessment of 3D tissue structures, i.e., surface morphology, nerve fascicles, nerve tissue volume, geometry, and vascular regrowth. The resulting data showed significant differences between operated animals and non-operated controls. COMPARISON WITH EXISTING METHODS Our approach avoids the sampling error associated with conventional 2D visualization approaches and holds promise for automation of the analysis of large series of datasets. CONCLUSIONS We have presented a potential way for 3D imaging and analysis of entire regenerated nerves non-destructively, paving the way for high-throughput analysis of therapeutic conditions of treating adult nerve injuries.