Multimodal optical characterisation of collagen photodegradation by femtosecond infrared laser ablation.

Collagen is a structural component of the human body, as a connective tissue it can become altered as a result of pathophysiological conditions. Although the collagen degradation mechanism is not fully understood, it plays an important role in ageing, disease progression and applications in therapeutic laser treatments. To fully understand the mechanism of collagen alteration, in our study photo-disruptive effects were induced in collagen I matrix by point-irradiation with a femtosecond Ti-sapphire laser under controlled laser ablation settings. This was followed by multi-modal imaging of the irradiated and surrounding areas to analyse the degradation mechanism. Our multi-modal methodology was based on second harmonic generation (SHG), scanning electron microscope (SEM), autofluorescence (AF) average intensities and the average fluorescence lifetime. This allowed us to quantitatively characterise the degraded area into four distinct zones: (1) depolymerised zone in the laser focal spot as indicated by the loss of SHG signal, (2) enhanced crosslinking zone in the inner boundary of the laser induced cavity as represented by the high fluorescence ring, (3) reduced crosslinking zone formed the outer boundary of the cavity as marked by the increased SHG signal and (4) native collagen. These identified distinct zones were in good agreement with the expected photochemical changes shown using Raman spectroscopy. In addition, imaging using polarisation-resolved SHG (p-SHG) revealed both a high degree of fibre re-orientation and a SHG change in tensor ratios around the irradiation spot. Our multi-modal optical imaging approach can provide a new methodology for defining distinct zones that can be used in a clinical setting to determine suitable thresholds for applying safe laser treatments without affecting the surrounding tissues. Furthermore this technique can be extended to address challenges observed in collagen based tissue engineering and used as a minimally invasive diagnostic tool to characterise diseased and non-diseased collagen rich tissues

Early evaluation of corneal collagen crosslinking in ex-vivo human corneas using two-photon imaging

The clinical outcome of corneal collagen crosslinking (CXL) is typically evaluated several weeks after treatment. An earlier assessment of its outcome could lead to an optimization of the treatment, including an immediate re-intervention in case of failure, thereby, avoiding additional discomfort and pain to the patient. In this study, we propose two-photon imaging (TPI) as an earlier evaluation method. CXL was performed in human corneas by application of riboflavin followed by UVA irradiation. Autofluorescence (AF) intensity and lifetime images were acquired using a commercial clinically certified multiphoton tomograph prior to CXL and after 2h, 24h, 72h, and 144h storage in culture medium. The first monitoring point was determined as the minimum time required for riboflavin clearance from the cornea. As control, untreated samples and samples treated only with riboflavin (without UVA irradiation) were monitored at the same time points. Significant increases in the stroma AF intensity and lifetime were observed as soon as 2h after treatment. A depth-dependent TPI analysis showed higher AF lifetimes anteriorly corresponding to areas were CXL was most effective. No alterations were observed in the control groups. Using TPI, the outcome of CXL can be assessed non-invasively and label-free much sooner than with conventional clinical devices.European Union Horizon 2020 (LASER-HISTO); European project FLIMVERTIC

Multimodal optical systems for clinical oncology

This thesis presents three multimodal optical (light-based) systems designed to improve the capabilities of existing optical modalities for cancer diagnostics and theranostics. Optical diagnostic and therapeutic modalities have seen tremendous success in improving the detection, monitoring, and treatment of cancer. For example, optical spectroscopies can accurately distinguish between healthy and diseased tissues, fluorescence imaging can light up tumours for surgical guidance, and laser systems can treat many epithelial cancers. However, despite these advances, prognoses for many cancers remain poor, positive margin rates following resection remain high, and visual inspection and palpation remain crucial for tumour detection. The synergistic combination of multiple optical modalities, as presented here, offers a promising solution. The first multimodal optical system (Chapter 3) combines Raman spectroscopic diagnostics with photodynamic therapy using a custom-built multimodal optical probe. Crucially, this system demonstrates the feasibility of nanoparticle-free theranostics, which could simplify the clinical translation of cancer theranostic systems without sacrificing diagnostic or therapeutic benefit. The second system (Chapter 4) applies computer vision to Raman spectroscopic diagnostics to achieve spatial spectroscopic diagnostics. It provides an augmented reality display of the surgical field-of-view, overlaying spatially co-registered spectroscopic diagnoses onto imaging data. This enables the translation of Raman spectroscopy from a 1D technique to a 2D diagnostic modality and overcomes the trade-off between diagnostic accuracy and field-of-view that has limited optical systems to date. The final system (Chapter 5) integrates fluorescence imaging and Raman spectroscopy for fluorescence-guided spatial spectroscopic diagnostics. This facilitates macroscopic tumour identification to guide accurate spectroscopic margin delineation, enabling the spectroscopic examination of suspicious lesions across large tissue areas. Together, these multimodal optical systems demonstrate that the integration of multiple optical modalities has potential to improve patient outcomes through enhanced tumour detection and precision-targeted therapies.Open Acces

Two-photon imaging of the cornea using femtosecond laser microscopes and tomographs

The cornea plays a crucial role on visual acuity. Diseases affecting this tissue are the second major cause of blindness worldwide. In clinical practice, it is commonly examined using methods that focus on the morphological analysis while the structural organization of the stroma and the cell’s metabolism are disregarded. Two-photon imaging (TPI) can provide these data. In this study, I demonstrate the advantages of TPI as a new imaging modality for the examination of the cornea. Two systems optimized for corneal imaging were used: a custom-built two-photon laser scanning microscope and a commercial multiphoton tomograph. Both systems were equipped with ultra-short near-infrared Ti:sapphire lasers. I show that TPI can provide a more complete assessment of the corneal state than the current clinical devices. Moreover, the potential of TPI for the following three clinical applications is shown: 1) it allows a better evaluation of human corneas in cornea banks which could improve sample selection prior to transplantation, 2) it enables the discrimination between non-pathological and diseased corneas based on the combined analysis of the tissue morphology, metabolism, and structural organization, and 3) it provides a faster evaluation of the outcome of corneal collagen crosslinking. The results indicate that, the multimodal analysis of function and morphology using a novel medical device based on TPI can lead to an improved corneal examination, thus improving diagnosis and care.Die Cornea spielt für die Sehschärfe eine entscheidende Rolle. Krankheiten, die dieses Gewebe befallen sind weltweit die zweitwichtigste Ursache für Blindheit. In der klinischen Praxis wird die Cornea gewöhnlich nur morphologisch untersucht, wobei strukturelle Zusammensetzung des Stromas und Zellmetabolismus nicht erfasst werden. Diese Informationen liefert die Zweiphotonen-Bildgebung (TPI). In der vorliegenden Arbeit zeige ich die Vorteile der TPI als neues bildgebendes Verfahren zur Cornea-Untersuchung. Dazu wurden zwei für die Korneabildgebung optimierte Systeme eingesetzt: Ein speziell angefertigtes Zwei-Photonen-Laserscanningmikroskop and ein kommerzieller Multiphotonen-Tomograph. Beide Systeme waren mit ultrakurzen-nahinfraroten Ti:Saphir-Lasern ausgestattet. Ich zeige, dass TPI eine vollständigere Beurteilung des Corneazustandes ermöglicht als herkömmliche klinische Geräte. Außerdem wird das TPI-Potential für drei klinische Anwendungen dargestellt: TPI ermöglicht 1) eine verbesserte Beurteilung humaner Spender-Corneae, was die Auswahl vor eine Transplantation verbessern könnte, 2) die Unterscheidung von gesunden und pathologischen Corneae basierend auf einer kombinierten Analyse von Morphologie, Metabolismus und strukturellem Aufbau, 3) eine schnellere Beurteilung des Effectes von Kollagen-Crosslinkings. Die Ergebnisse zeigen, dass eine multimodale Auswertung von Funktion und Morphologie zu einer verbesserten Cornea-Untersuchung und dadurch verbesserter Diagnose und Behandlung führen kann

TECHNART 2017. Non-destructive and microanalytical techniques in art and cultural heritage. Book of abstracts

440 p.TECHNART2017 is the international biannual congress on the application of Analytical Techniques in Art and Cultural Heritage. The aim of this European conference is to provide a scientific forum to present and promote the use of analytical spectroscopic techniques in cultural heritage on a worldwide scale to stimulate contacts and exchange experiences, making a bridge between science and art. This conference builds on the momentum of the previous TECHNART editions of Lisbon, Athens, Berlin, Amsterdam and Catania, offering an outstanding and unique opportunity for exchanging knowledge on leading edge developments. Cultural heritage studies are interpreted in a broad sense, including pigments, stones, metal, glass, ceramics, chemometrics on artwork studies, resins, fibers, forensic applications in art, history, archaeology and conservation science. The meeting is focused in different aspects: - X-ray analysis (XRF, PIXE, XRD, SEM-EDX). - Confocal X-ray microscopy (3D Micro-XRF, 3D Micro-PIXE). - Synchrotron, ion beam and neutron based techniques/instrumentation. - FT-IR and Raman spectroscopy. - UV-Vis and NIR absorption/reflectance and fluorescence. - Laser-based analytical techniques (LIBS, etc.). - Magnetic resonance techniques. - Chromatography (GC, HPLC) and mass spectrometry. - Optical imaging and coherence techniques. - Mobile spectrometry and remote sensing

Properties and Applications of Graphene and Its Derivatives

Graphene is a two-dimensional, one-atom-thick material made entirely of carbon atoms, arranged in a honeycomb lattice. Because of its distinctive mechanical (e.g., high strength and flexibility) and electronic (great electrical and thermal conductivities) properties, graphene is an ideal candidate in myriad applications. Thus, it has just begun to be engineered in electronics, photonics, biomedicine, and polymer-based composites, to name a few. The broad family of graphene nanomaterials (including graphene nanoplatelets, graphene oxide, graphene quantum dots, and many more) go beyond and aim higher than mere single-layer (‘pristine’) graphene, and thus, their potential has sparked the current Special Issue. In it, 18 contributions (comprising 14 research articles and 4 reviews) have portrayed probably the most interesting lines as regards future and tangible uses of graphene derivatives. Ultimately, understanding the properties of the graphene family of nanomaterials is crucial for developing advanced applications to solve important challenges in critical areas such as energy and health

Green synthesis and characterization of gold nanoparticles from South African plants and their biological evaluations

Philosophiae Doctor - PhDThe field of nanotechnology continues to offer solutions for biotechnologists whose target is to improve the quality of life by finding new therapies to combat diseases. Gold nanoparticles (AuNPs) have been showing great potentials in many biomedical applications. The antibacterial activity of the AuNPs presents a therapeutic option for conditions caused by bacterial infections such as chronic wounds. Also, these versatile particles can offer solutions in the treatments of infectious diseases and can also be exploited as “smart” vehicles to carry drugs, such as antibiotics, for improved efficiency. Moreover, the anti-inflammatory activity of AuNPs makes them useful in the management of prolonged inflammation caused by bacterial infections. The synthesis of AuNPs can be achieved by variety of physical and chemical methods that have been successfully applied in labs and industry. Nonetheless, the drawbacks of these “conventional” methods in terms of high cost, adverse health side effects and incompatibility with the ecosystem cannot be overlooked. Thus, new safer and more cost-effective protocols have been reported for the synthesis of AuNPs. Plants have provided alternate synthesis methods in which the reducing capabilities of the phytochemicals, found in the aqueous plant extracts, can be used to chemically synthesize AuNPs from gold precursors. The biosynthesis and characterization of AuNPs from the phytochemicals of several South African plants is investigated in this study. The study also reports the optimization of the AuNPs biosynthesis by varying reaction conditions such as temperature and plant extracts’ concentrations. Furthermore, the study highlights the wound healing activity of the AuNPs synthesized from selected plants by investigating their antibacterial activity on bacterial strains known to cause chronic wounds. The ability of these AuNPs to carry ampicillin in order to enhance the antibacterial activity is also described herein. The cytotoxicity of the biosynthesized AuNPs was evaluated on human normal fibroblasts cells (KMST-6). Additionally, the immunomodulatory effect of the biosynthesized AuNPs on the cytokines production from macrophages and Natural Killer (NK) cells was examined. The study was successful to produce biocompatible and safe AuNPs synthesized from the tested aqueous plant extracts. The resulted AuNPs showed different physicochemical properties by varying the reaction conditions. The AuNPs exhibited antibacterial activity against several Gram-positive and Gram-negative bacteria. Also, ampicillin was successfully loaded on the biosynthesized AuNPs, which led to the formation of more antibacterial active conjugated AuNPs compared to the free AuNPs. The green synthesized AuNPs were also found to have anti-inflammatory responses as shown by the reduction of pro-inflammatory cytokines from immune cells. In vitro assays showed that the biogenic AuNPs were not toxic to KMST-6 cells. Overall, the data suggest that plant extracts produce biologically safe AuNPs with antibacterial and anti-inflammatory activities that can be exploited in the treatment of chronic wounds and in the management of chronic inflammation

Polymer-Coated Inorganic Nanoparticles: Nanotools for Life Science Applications

This dissertaion focus on the synthesis, surface modification and characterization of inorganic nanoparticles(NPs), including magnetic, plasmonic and semiconductor NPs. With controlling the reaction conditions during the synthesis, different particle diameters in the range of 4 nm to 30 nm can be synthesized. Afterwards, polymer coating process was successfully applied to different materials by overcoating the NPs with an amphiphoilic polymer, which can make the particle water soluble. This work aimed to produce the polymer-­ coated nanoparticles,analyze and compare their physico-­‐chemical properties based on different materials,and further, to test their potential for different biological applications

Vat 3D printable materials and post-3D printing procedures for the development of engineered devices for the biomedical field

L'abstract è presente nell'allegato / the abstract is in the attachmen