Nanostructure-specific X-ray tomography reveals myelin levels, integrity and axon orientations in mouse and human nervous tissue

Myelin insulates neuronal axons and enables fast signal transmission, constituting a key component of brain development, aging and disease. Yet, myelin-specific imaging of macroscopic samples remains a challenge. Here, we exploit myelin’s nanostructural periodicity, and use small-angle X-ray scattering tensor tomography (SAXS-TT) to simultaneously quantify myelin levels, nanostructural integrity and axon orientations in nervous tissue. Proof-of-principle is demonstrated in whole mouse brain, mouse spinal cord and human white and gray matter samples. Outcomes are validated by 2D/3D histology and compared to MRI measurements sensitive to myelin and axon orientations. Specificity to nanostructure is exemplified by concomitantly imaging different myelin types with distinct periodicities. Finally, we illustrate the method’s sensitivity towards myelin-related diseases by quantifying myelin alterations in dysmyelinated mouse brain. This non-destructive, stain-free molecular imaging approach enables quantitative studies of myelination within and across samples during development, aging, disease and treatment, and is applicable to other ordered biomolecules or nanostructures

High-speed tensor tomography: iterative reconstruction tensor tomography (IRTT) algorithm

The recent advent of tensor tomography techniques has enabled tomographic investigations of the 3D nanostructure organization of biological and material science samples. These techniques extended the concept of conventional X-ray tomography by reconstructing not only a scalar value such as the attenuation coefficient per voxel, but also a set of parameters that capture the local anisotropy of nanostructures within every voxel of the sample. Tensor tomography data sets are intrinsically large as each pixel of a conventional X-ray projection is substituted by a scattering pattern, and projections have to be recorded at different sample angular orientations with several tilts of the rotation axis with respect to the X-ray propagation direction. Currently available reconstruction approaches for such large data sets are computationally expensive. Here, a novel, fast reconstruction algorithm, named iterative reconstruction tensor tomography (IRTT), is presented to simplify and accelerate tensor tomography reconstructions. IRTT is based on a second-rank tensor model to describe the anisotropy of the nanostructure in every voxel and on an iterative error backpropagation reconstruction algorithm to achieve high convergence speed. The feasibility and accuracy of IRTT are demonstrated by reconstructing the nanostructure anisotropy of three samples: a carbon fiber knot, a human bone trabecula specimen and a fixed mouse brain. Results and reconstruction speed were compared with those obtained by the small-angle scattering tensor tomography (SASTT) reconstruction method introduced by Liebi et al. [Nature (2015), 527, 349–352]. The principal orientation of the nanostructure within each voxel revealed a high level of agreement between the two methods. Yet, for identical data sets and computer hardware used, IRTT was shown to be more than an order of magnitude faster. IRTT was found to yield robust results, it does not require prior knowledge of the sample for initializing parameters, and can be used in cases where simple anisotropy metrics are sufficient, i.e. the tensor approximation adequately captures the level of anisotropy and the dominant orientation within a voxel. In addition, by greatly accelerating the reconstruction, IRTT is particularly suitable for handling large tomographic data sets of samples with internal structure or as a real-time analysis tool during the experiment for online feedback during data acquisition. Alternatively, the IRTT results might be used as an initial guess for models capturing a higher complexity of structural anisotropy such as spherical harmonics based SASTT in Liebi et al. (2015), improving both overall convergence speed and robustness of the reconstruction

Design and development of new nanosystem based on self-assembly peptides for nanomedicine applications

Peptide-based materials (PBMs) generate by the aggregation of amphiphilic monomers represent a rapidly growing tool within materials science. They have been considered for several applications in different fields from electronic to nanomedicine. According to an appropriate design, amphiphilic peptides can spontaneously assemble in well-structured supramolecular materials as result of an intricate network of inter- and/or intra-molecular interactions between hydrophobic and hydrophilic portions. The interaction manner can strongly influence both morphology and properties of the final supramolecular materials. Aromaticity is the terms used to describe the particular molecular stability associated to a cyclic shaped and planar chemical entity with a ring of resonance bonds. The peculiar electronic structure of aromatics molecules arouses their interesting physicochemical properties. All the classes of intermolecular forces known as the aromatic interaction give raise from it. The knowledge and the study of aromatic interaction contaminated peptide based material field too. To this day, using self-assembly as instrument of bottom-up nano-plenning, it is a clear evidence that aromatic peptide sequences, containing phenylalanine (Phe), tyrosine (Tyr) or tryptophan (Trp), can be opportunely modulated to the aim of generate the required supramolecular architecture. The forefather of this new class of peptide monomers is the well-known diphenylalanine (FF) homopeptide. Through a combination of π-π staking and hydrogen-bonding, different method of preparation, specific pH values or solvent, FF is able to self-organize in different kinds of nano- and macro- morphologies (hollow nanotubes, fibers, vesicles, metastable hydrogels or organogels). By this evidence, many structural analogues of diphenylalanine were studied and peptide nanostructures containing the FF motif or more extended aromatic sequences have been investigated for their mechanical, electrochemical and optical properties, and more recently for some nanomedicine applications. Nevertheless, the majority of studies reported in literature are principally focused on clarifying the physicochemical aspects responsible for array stability in FF based nanostructures, whereas only few studies have been devoted in the investigation of FF aggregates for biomedical applications. This essentially because of the intrinsic low water solubility of these peptide sequences. According to these considerations, during the three years of the PhD project, novel poly-phenylalanine self-assembling conjugates were carefully designed, synthetized and fully characterized. The final peptide materials were evaluated for potential applications in bio-imaging field (with particular bearing to Magnetic Resonance Imaging and fluorescence imaging). It was also appreciate as the chemical modification of the aromatic framework with chelating agents, gadolinium (Gd) complexes, and polyethylene glycol (PEG) fragments with different length can affect the structural organization and the supramolecular behavior of the nanomaterial. The result produced on the hierarchical organization by the chemical replacement of the Phe with others aromatic amino acids (such as tryptophan, tyrosine and 2-naphthylalanine) was also investigated. The entirety of collected data during this PhD project permits to highlight the possible relationship existing between the chemical structure of the proposed building blocks, the final supramolecular nanostructure, and their functional features. In order to simplify the comprehension and the discussion of the results (Chapter III), they will be argued in three separate sections: • Section I: PBMs as supramolecular contrast agents for MRI. • Section II: PBMs as photoluminescent supramolecular probes. • Section III: PBMs obtained by punctual chemical modifications of homophenylalanine sequences. In Section I, an innovative class of supramolecular CAs for MRI, based on peptide self-assembly monomers, is described and analyzed. Different design strategies were applied to obtain and improve the aggregation phenomenon. The structural and relaxometric properties of each self-assembling system are discussed and mutually compared. The improved values of relaxivity and the exanimated capability to encapsulate the doxorubicin anticancer drug suggest a potential use of the proposed nanostructures as new theranostic platform. Photoluminescent (PL) phenomena in peptide-based materials are the subject of Section II. A class of novel PEGylated homo-phenylalanine was synthetized and, due to the high content of β-sheet, the final self-assembled systems show blue PL emission. A red-shift of the fluorescence was actualized by FRET phenomena between the nanostructure and a internalized NBD dye. Committing to the hydrogen bonding hypothesis, a relationship between observed PL and the number of interaction sites in nanostructures was developed. In Section III the effect of a punctual chemical modification on peptide primary sequence will be elucidated. Hetero- and homo-sequences were derived simply by the replacement of Phe residues with tyrosine and tryptophan ones. Characteristic gelification behavior was found for peptides containing Tyr. WAXS/SAXS studies and molecular dynamic simulations supported the structural analysis of the new peptide-based materials. The experimental protocols are totally collected in the dedicated Experimental section. The full characterization of synthetized peptides, conjugates and derivatives is reported in Appendix I, meanwhile additional information, Tables and Figures are gather together in Appendix II

A multi-scale imaging approach to understand osteoarthritis development

X-ray phase-contrast imaging is an innovative and advanced imaging method. Contrary to conventional radiology, where the image contrast is primarily determined by X-ray attenuation, phase-contrast images contain additional information generated by the phase shifts or refraction of the X-rays passing through matter. The refractive effect on tissue samples is orders of magnitude higher than the absorption effect in the X-ray energy range used in biomedical imaging. This technique makes it possible to produce excellent and enhanced image contrast, particularly when examining soft biological tissues or features with similar X-ray attenuation properties. In combination with high spatial resolution detector technology and computer tomography, X-ray phase-contrast imaging has been proved to be a powerful method to examine tissue morphology and the evolution of pathologies three-dimensionally, with great detail and without the need of contrast agents. This Thesis work has focused on developing an accurate, multi-scale X-ray-based methodology for imaging and characterizing the early stages of osteoarthritis. X-ray phase-contrast images acquired at different spatial resolutions provide unprecedented insights into cartilage and the development of its degeneration, i.e., osteoarthritis. Other types of X-ray phase-contrast imaging techniques and setups using spatial resolutions ranging from micrometer down to nanometer were applied. Lower spatial resolutions allow large sample coverage and comprehensive representations, while the nanoscale analysis provides a precise depiction of anatomical details and pathological signs. X-ray phase-contrast results are correlated to data obtained, on the same specimens, by standard laboratory methods, such as histology and transmission electron microscopy. Furthermore, X-ray phase-contrast images of cartilage were acquired using different X-ray sources and results were compared in terms of image quality. It was shown that with the use of synchrotron radiation, more detailed images and much faster data acquisitions could be achieved. A second focus in this Thesis work has been the investigation of the reaction of healthy and degenerated cartilage under different physical pressures, simulating the different levels of stress to which the tissue is subject during daily movements. A specifically designed setup was used to dynamically study cartilage response to varying pressures with X-ray phase-contrast micro-computed tomography, and a fully volumetric and quantitative methodology to accurately describe the tissue morphological variations. This study revealed changes in the behavior of the cartilage cell structure, which differ between normal and osteoarthritic cartilage tissues. The third focus of this Thesis is the realization of an automated evaluation procedure for the discrimination of healthy and cartilage images with osteoarthritis. In recent years, developments in neural networks have shown that they are excellently suited for image classification tasks. The transfer learning method was applied, in which a pre-trained neural network with cartilage images is further trained and then used for classification. This enables a fast, robust and automated grouping of images with pathological findings. A neural network constructed in this way could be used as a supporting instrument in pathology. X-ray phase-contrast imaging computed tomography can provide a powerful tool for a fully 3D, highly accurate and quantitative depiction and characterization of healthy and early stage-osteoarthritic cartilage, supporting the understanding of the development of osteoarthritis.Röntgen-Phasenkontrast-Bildgebung ist eine innovative und weiterführende Bildgebungsmethode. Im Gegensatz zu herkömlichen Absorptions-Röntgenaufnahmen, wie sie in der Radiologie verwendet werden, wird der Kontrast bei dieser Methode aus dem Effekt der Phasenverschiebung oder auch Brechung der Röngtenstrahlen gebildet. Der Brechungseffekt bei Gewebeproben ist um ein Vielfaches höher als der Absorptionseffekt des elektromagnetischen Spektrums der Röntgenstrahlen. Diese Methode ermöglicht die Darstellung von großen Kontraste im Gewebe. Unter Verwendung eines hochauflösenden Detektors und in Kombination mit der Computer-Tomographie, ist Phasenkontrast-Bildgebung eine sehr gute Methode um Knorpelgewebe und Arthrose im Knorpel zu untersuchen. Diese Arbeit beschreibt primär ein Verfahren zur Darstellung arthrotischen Knorpels im Anfangsstadium. Die mit verschiedenen Auflösungen und 3D-Phasen-Kontrast-Methoden produzierten Aufnahmen ermöglichen einen noch nie dagewesenen Einblick in den Knorpel und die Entwicklung von Arthrose im Anfangsstadium. Hierbei kam die propagationsbasierte Phasenkontrastmethode mit einer Auflösung im mikrometer Bereich und die (Nano)-Holotomographie-Methode mit einer Auflösung im Submicrometer Bereich zum Einsatz. Durch Auflösung im mikrometer Bereich kann ein großes Volumen im Knorpel gescannt werden, während die Nano-Holotomographie Methode eine sehr große Detailauflösung aufweißt. Die Phasenkontrast-Aufnahmen werden mit zwei anderen wissenschaftlichen Methoden verglichen: mikroskopische Abbildungen histologisch aufgearbeiteter Knorpelproben und Aufnahmen eines Transmissionselektroskop zeigen sehr große Übereinstimmungen zur Röntgen-Phasenkontrast-Bildgebung. Desweiteren wurden Phasenkontrast-Aufnahmen von Knorpel aus unterschiedlichen Röntgenquellen verglichen. Hierbei zeigte sich, dass mit Hilfe des Teilchenbeschleunigers (Synchrotron) detailreichere und schnellere Aufnahmen erzielt werden können. Bilder aus Flüssig-Metall-Quellen zeigen sich durchaus von guter Qualität, erfordern jedoch sehr lange Aufnahmezeiten. In dieser Arbeit wird zudem das Verhalten von Knorpelgewebe, welches ein Anfangsstadium von Arthrose aufweist, unter physikalischem Druck untersucht. Hierfür wurden 3D-Computertomographie-Aufnahmen von komprimiertem Knorpelgewebe angefertig und mit Aufnahmen ohne Komprimierung verglichen. Ein quantitativer Vergleich machte Veränderungen des Verhaltens der Knorpelzellstruktur (Chondronen) sichtbar. Es konnte gezeigt werden, dass Chondrone bei arthrotischem Knorpel ein verändertes Kompressionsverhalten haben. Der dritte Fokus dieser Arbeit liegt auf der automatisierten Auswertung von Aufnahmen gesunden und arthrotischen Knorpelgewebes. Die Entwicklungen im Bereich der Neuronale Netze zeigten in den letzten Jahren, dass diese sich hervoragend für Bildklassifizierungsaufgaben eignen. Es wurde die Methode des transferierenden Lernens angewandt, bei der ein vortrainiertes Neuronales Netz mit Knorpelbildern weitertrainiert und anschließend zur Klassifizierung eingesetzt wird. Dadurch ist eine schnelle, robuste und automatisierte Gruppierung von Bildern mit pathologischen Befunden möglich. Ein derart konstruiertes Neuronales Netz könnte als unterstützendes Instrument in der Pathologie angewandt werden. Röntgen-Phasenkontrast-CT kann ein leistungsstarkes Werkzeug für eine umfassende, hochpräzise und quantitative 3D-Darstellung und Charakterisierung von gesundem Knorpel und athrotischem Knorpel im Frühstadium bieten, um das Verständnis der Entwicklung von Osteoarthritis zu erweitern

Intrinsically Disordered Proteins and Chronic Diseases

This book is an embodiment of a series of articles that were published as part of a Special Issue of Biomolecules. It is dedicated to exploring the role of intrinsically disordered proteins (IDPs) in various chronic diseases. The main goal of the articles is to describe recent progress in elucidating the mechanisms by which IDPs cause various human diseases, such as cancer, cardiovascular disease, amyloidosis, neurodegenerative diseases, diabetes, and genetic diseases, to name a few. Contributed by leading investigators in the field, this compendium serves as a valuable resource for researchers, clinicians as well as postdoctoral fellows and graduate student

Microscopy Conference 2021 (MC 2021) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2021"

National synchrotron light source. Activity report, October 1, 1995--September 30, 1996

The hard work done by the synchrotron radiation community, in collaboration with all those using large-scale central facilities during 1995, paid off in FY 1996 through the DOE`s Presidential Scientific Facilities Initiative. In comparison with the other DOE synchrotron radiation facilities, the National Synchrotron Light Source benefited least in operating budgets because it was unable to increase running time beyond 100%-nevertheless, the number of station hours was maintained. The major thrust at Brookhaven came from a 15% increase in budget which allowed the recruitment of seven staff in the beamlines support group and permitted a step increment in the funding of the extremely long list of upgrades; both to the sources and to the beamlines. During the December 1995 shutdown, the VUV Ring quadrant around U10-U12 was totally reconstructed. New front ends, enabling apertures up to 90 mrad on U10 and U12, were installed. During the year new PRTs were in formation for the infrared beamlines, encouraged by the investment the lab was able to commit from the initiative funds and by awards from the Scientific Facilities Initiative. A new PRT, specifically for small and wide angle x-ray scattering from polymers, will start work on X27C in FY 1997 and existing PRTs on X26C and X9B working on macromolecular crystallography will be joined by new members. Plans to replace aging radio frequency cavities by an improved design, originally a painfully slow six or eight year project, were brought forward so that the first pair of cavities (half of the project for the X-Ray Ring) will now be installed in FY 1997. Current upgrades to 350 mA initially and to 438 mA later in the X-Ray Ring were set aside due to lack of funds for the necessary thermally robust beryllium windows. The Scientific Facilities Initiative allowed purchase of all 34 windows in FY 1996 so that the power upgrade will be achieved in FY 1997