3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described ...

Technical University of Munich

X-ray computed tomography (CT) is a powerful noninvasive technique for investigating the inner structure of objects and organisms. However, the resolution of laboratory CT systems is typically limited to the micrometer range. In this paper, we present a table-top nanoCT system in conjunction with standard processing tools that is able to routinely reach resolutions down to 100 nm without using X-ray optics. We demonstrate its potential for biological investigations by imaging a walking appendage of , a representative of Onychophora-an invertebrate group pivotal for understanding animal evolution. Comparative analyses proved that the nanoCT can depict the external morphology of the limb with an image quality similar to scanning electron microscopy, while simultaneously visualizing internal muscular structures at higher resolutions than confocal laser scanning microscopy. The obtained nanoCT data revealed hitherto unknown aspects of the onychophoran limb musculature, enabling the 3D reconstruction of individual muscle fibers, which was previously impossible using any laboratory-based imaging technique.

Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography.

Chemical staining of soft-tissues can be used as a strategy to increase their low inherent contrast in X-ray absorption micro-computed tomography (micro-CT), allowing to obtain fast three-dimensional structural information of animal organs. Though some staining agents are commonly used in this context, little is known about the staining agents' ability to stain specific types of tissues; the times necessary to provide a sufficient contrast; and the effect of staining solution in distorting the tissue. Here we contribute to studies of animal organs (mouse heart and lungs) using staining combined with dual-energy micro-CT (DECT). DECT was used in order to obtain an additional quantitative measure for the amount of staining agents within the sample in 3D maps. Our results show that the two staining solutions used in this work diffuse differently in the tissues studied, the staining times of some tens of minutes already produce high-quality micro-CT images and, at the concentrations applied in this work, the staining solutions tested do not cause relevant tissue distortions. While one staining solution provides images of the general morphology of the organs, the other reveals organs' features in the order of a hundred micrometers.

Dual-energy micro-CT for quantifying the time-course and staining characteristics of ex-vivo animal organs treated with iodine- and gadolinium-based contrast agents.

Many histological methods require staining of the cytoplasm, which provides instrumental details for diagnosis. One major limitation is the production of 2D images obtained by destructive preparation of 3D tissue samples. X-ray absorption micro- and nanocomputed tomography (microCT and nanoCT) allows for a nondestructive investigation of a 3D tissue sample, and thus aids to determine regions of interest for further histological examinations. However, application of microCT and nanoCT to biological samples (e.g., biopsies) is limited by the missing contrast within soft tissue, which is important to visualize morphological details. We describe an eosin-based preparation overcoming the challenges of contrast enhancement and selectivity for certain tissues. The eosin-based staining protocol is suitable for whole-organ staining, which then enables high-resolution microCT imaging of whole organs and nanoCT imaging of smaller tissue pieces retrieved from the original sample. Our results demonstrate suitability of the eosin-based staining method for diagnostic screening of 3D tissue samples without impeding further diagnostics through histological methods.

Three-dimensional virtual histology enabled through cytoplasm-specific X-ray stain for microscopic and nanoscopic computed tomography.

Grating-based phase-contrast computed tomography (GBPC-CT) enables increased soft tissue differentiation, but often suffers from streak artifacts when performing high-sensitivity GBPC-CT of biomedical samples. Current GBPC-CT setups consist of one-dimensional gratings and hence allow to measure only the differential phase-contrast (DPC) signal perpendicular to the direction of the grating lines. Having access to the full two-dimensional DPC signal can strongly reduce streak artefacts showing up as characteristic horizontal lines in the reconstructed images. GBPC-CT with gratings tilted by 45° around the optical axis, combining opposed projections, and reconstructing with filtered backprojection is one method to retrieve the full three-dimensional DPC signal. This approach improves the quality of the tomographic data as already demonstrated at a synchrotron facility. However, additional processing and interpolation is necessary, and the approach fails when dealing with cone-beam geometry setups. In this work, we employ the tilted grating configuration with a laboratory GBPC-CT setup with cone-beam geometry and use statistical iterative reconstruction (SIR) with a forward model accounting for diagonal grating alignment. Our results show a strong reduction of streak artefacts and significant increase in image quality. In contrast to the prior approach our proposed method can be used in a laboratory environment due to its cone-beam compatibility.

Tilted grating phase-contrast computed tomography using statistical iterative reconstruction.

Dual-energy CT has opened up a new level of quantitative X-ray imaging for many diagnostic applications. The energy dependence of the X-ray attenuation is the key to quantitative material decomposition of the volume under investigation. This material decomposition allows the calculation of virtual native images in contrast enhanced angiography, virtual monoenergetic images for beam-hardening artifact reduction and quantitative material maps, among others. These visualizations have been proven beneficial for various diagnostic questions. Here, we demonstrate a new method of 'virtual dual-energy CT' employing grating-based phase-contrast for quantitative material decomposition. Analogue to the measurement at two different energies, the applied phase-contrast measurement approach yields dual information in form of a phase-shift and an attenuation image. Based on these two image channels, all known dual-energy applications can be demonstrated with our technique. While still in a preclinical state, the method features the important advantages of direct access to the electron density via the phase image, simultaneous availability of the conventional attenuation image at the full energy spectrum and therefore inherently registered image channels. The transfer of this signal extraction approach to phase-contrast data multiplies the diagnostic information gained within a single CT acquisition. The method is demonstrated with a phantom consisting of exemplary solid and fluid materials as well as a chicken heart with an iodine filled tube simulating a vessel. For this first demonstration all measurements have been conducted at a compact laser-undulator synchrotron X-ray source with a tunable X-ray energy and a narrow spectral bandwidth, to validate the quantitativeness of the processing approach.

Direct quantitative material decomposition employing grating-based X-ray phase-contrast CT.

Propagation-based phase-contrast computed tomography has become a valuable tool for visualization of three-dimensional biological samples, due to its high contrast between materials with similar attenuation properties. However, one of the most-widely used phase-retrieval algorithms imposes a homogeneity assumption onto the sample, which leads to artifacts for numerous applications where this assumption is violated. Prominent examples are biological samples with highly-absorbing implants. Using synchrotron radiation, we demonstrate by the example of a guinea pig inner ear with a cochlear implant electrode, how a recently developed model-based iterative algorithm for propagation-based phase-contrast computed tomography yields distinct benefits for such a task. We find that the model-based approach improves the overall image quality, removes the detrimental influence of the implant and accurately visualizes the cochlea.

Propagation-based phase-contrast tomography of a guinea pig inner ear with cochlear implant using a model-based iterative reconstruction algorithm.

Histological investigations are indispensable with regards to the identification of structural tissue details but are limited to two-dimensional images, which are often visualized in one and the same plane for comparison reasons. Nondestructive three-dimensional technologies such as X-ray micro- and nanoCT have proven to provide valuable benefits for the understanding of anatomical structures as they allow visualization of structural details in 3D and from arbitrary viewing angles. Nevertheless, low attenuation of soft tissue has hampered their application in the field of 3D virtual histology. We present a hematein-based X-ray staining method that specifically targets the cell nuclei of cells, as demonstrated for a whole liver lobule of a mouse. Combining the novel staining protocol with the high resolving power of a recently developed nanoCT system enables the 3D visualization of tissue architecture in the nanometer range, thereby revealing the real 3D morphology and spatial distribution of the cell nuclei. Furthermore, our technique is compatible with conventional histology, as microscopic slides can be derived from the very same stained soft-tissue sample and further counter staining is possible. Thus, our methodology demonstrates future applicability for modern histopathology using laboratory X-ray CT devices.

Nucleus-specific X-ray stain for 3D virtual histology.

As iterative reconstruction in Computed Tomography (CT) is an ill-posed problem, additional prior information has to be used to get a physically meaningful result (close to ground truth if available). However, the amount of influence of the regularisation prior is crucial to the outcome of the reconstruction. Therefore, we propose a scheme for tuning the strength of the prior via a certain image metric. In this work, the parameter is tuned for minimal histogram entropy in selected regions of the reconstruction as histogram entropy is a very basic approach to characterise the information content of data. We performed a sweep over different regularisation parameters showing that the histogram entropy is a suitable metric as it is well behaved over a wide range of parameters. The parameter determination is a feedback loop approach we applied to numerically simulated FORBILD phantom data and verified with an experimental measurement of a micro-CT device. The outcome is evaluated visually and quantitatively by means of root mean squared error (RMSE) and structural similarity (SSIM) for the simulation and visually for the measured sample (no ground truth available). The final reconstructed images exhibit noise-suppressed iterative reconstruction. For both datasets, the optimisation is robust where its initial value is concerned. The parameter tuning approach shows that the proposed metric-driven feedback loop is a promising tool for finding a suitable regularisation parameter in statistical iterative reconstruction.

Metric-guided regularisation parameter selection for statistical iterative reconstruction in computed tomography.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Author Correction: Nucleus-specific X-ray stain for 3D virtual histology.

Tardigrades (water bears) are microscopic invertebrates of which the anatomy has been well studied using traditional techniques, but a comprehensive three-dimensional reconstruction has never been performed. In order to close this gap, we employed X-ray computed tomography (CT), a technique that is becoming increasingly popular in zoology for producing high-resolution, three-dimensional (3D) scans of whole specimens. While CT has long been used to scan larger samples, its use in some microscopic animals can be problematic, as they are often too small for conventional CT yet too large for high-resolution, optics-based soft X-ray microscopy. This size gap continues to be narrowed with advancements in technology, with high-resolution imaging now being possible using both large synchrotron devices and, more recently, laboratory-based instruments.

Sebastian Allner

Department of Physics and Munich School of BioEngineering

Technical University of Munich

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

Sebastian Allner

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physics and Munich School of BioEngineering

Technical University of Munich

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

Department of Physics and Munich School of BioEngineering