Дисертації з теми "Microfluidic light scattering blood"

This dissertation focused on detecting waterborne pathogens in a microfluidic biosensing system which enables point-of-care, real-time monitoring. Within this framework, I have been addressing three objectives. The first objective was to enhance mixing of particles in a microfluidic device. To this end, SDS (sodium dodecyl sulfate) or Tween 80 (polyethylene sorbitol ester) was added to the antibody-conjugated polystyrene microparticle suspension. Both surfactants showed non-specific binding (with SDS) or very poor diffusion (with Tween 80). As an alternative approach, highly carboxylated polystyrene microparticles showed very low non-specific binding comparable to that with Tween 80 and good diffusional mixing equivalent to that with SDS. This work was published in Appendix A (© 2008 Elsevier). The second objective was to detect E. coli K-12 using the microfluidic-based system with low detection limit in Appendix B (© 2008 Elsevier). This method was essentially one-step and requires no sample pre-treatment or cell culturing. Conventional immunoassay using polyclonal antibody detects not only viable cells, but also dead cell and free antigens. In order to reduce false positive signal originated from dead cells and free antigens, target solution was washed three times. The detection limit was as low as 40 cfu ml⁻¹ or 4 cfu per device (viable cells only), or <10 cfu ml⁻¹ or <1 cfu per device (including dead cells and free antigens). Our final objective was to develop real-time, high sensitive method for detecting waterborne pathogens in a water distribution system in Appendix C. Detection of Escherichia coli (E. coli) in a single straight pipe was demonstrated using a microfluidic system utilizing light scattering detection of latex immunoagglutination assay. Assay time is <5 min per assay and detection limit is 10 cfu ml⁻¹. Optical signals are compared with viable E. coli counts (not real time) and salt tracer experiments. Laminar (Re = 1,102) and turbulent (Re = 6,144) flows are used to simulate the flow regimes in a real water distribution system.

This dissertation develops technology for microfluidic point-of-care immunoassay devices. This research (2004–2007) improved microfluidic immunoassay performance by reducing reagent consumption, decreasing analysis time, increasing sensitivity, and integrating processes using a lab-on-a-chip. Estimates show that typical hospital laboratories can save $1.0 million per year by using microfluidic chips. Our first objective was to enhance mixing in a microfluidic channel, which had been one of the main barriers to using these devices. Another goal of our studies was to simplify immunoassays by eliminating surfactants. Manufacturers of latex immunoassays add surfactants to prevent non-specific aggregation of microspheres. However, these same surfactants can cause false positives (and negatives) during diagnostic testing. This work, published in Appendix A (© 2006 Elsevier) shows that highly carboxylated polystyrene (HCPS) microspheres can replace surfactants and induce rapid mixing via diffusion in microfluidic devices. Our second objective was to develop a microfluidic device using fiber optics to detect static light scattering (SLS) of microspheres in Appendix B (© 2007 Elsevier). Fiber optics were used to deliver light emitting diode (LED) or laser light. A miniature spectrometer was used to measure 45° forward light scattering collected by optical fiber. Latex microspheres coated with PR3 proteins were used to test for the vasculitis marker, anti-PR3. No false negatives or positives were observed. A limit of detection (LOD) of 50 ng mL⁻¹ was demonstrated. This optical detection system works without fluorescence or chemiluminescence markers. It is cost effective, small, and re-usable with simple rinsing. The final objective in this dissertation, published in Appendix C (© 2007 Elsevier), developed a multiplex immunoassay. A lab-on-a-chip was used to detect multiple antibodies using microsphere light scattering and quantum dot (QD) emission. We conjugated QDs onto microspheres and named this configuration “nano-on-micro” or “NOM”. Upon radiation with UV light, strong light scattering is observed. Since QDs also provide fluorescent emission, we are able to use increased light scattering for detecting antigen-antibody reactions, and decreased QD emission to identify which antibody is present.

Abstract Optical coherence tomography (OCT) is a modern rapidly developing technique for non-invasive imaging of the internal structure of optically non-uniform objects based on the principles of low-coherent interferometry. However, multiple scattering of light in the objects under study brings distortions to the images obtained by OCT. The analysis of formation of the OCT signals is required for understanding the role of multiple scattering in this formation and providing recommendations for optimal configuration of a measuring setup. In the present thesis formation of the OCT signals and images is analyzed implementing Monte Carlo simulations of light propagation in scattering media. Blood, intralipid solution, human skin and paper samples are chosen as the objects under study due to the interest in the diagnostics of these objects in biomedicine and paper industry. Multilayer models of skin phantoms, skin and paper were developed in the frames of the present study for simulation of OCT signals and two-dimensional OCT images of these objects. The contribution of different scattering orders as well as different fractions of photons (least and multiply scattered, diffusive and non-diffusive) to these images was found allowing to evaluate the maximal depth of non-distorted imaging in each particular case. The simulated OCT images were compared to the experimental ones demonstrating qualitative similarity. This fact allowed the author to analyze qualitatively the influence of parameters of the OCT setup on the images which have also been acquired in this work. The formation of the OCT images of paper samples with various refractive index matching liquids was also studied.

Master of Science
Department of Chemistry
Christopher T. Culbertson
Circulating tumor cells (CTCs) have become a key component in the identification and treatment of cancer. Once dislodged from the main tumor, CTCs travel through the bloodstream and cause metastasis. Early detection and identification of these cells can help in the evaluation and prognosis of various types of cancer, as well as assisting in patient treatments by determining the spread of the disease. Here, a high-throughput microfluidic analysis technique is described that can efficiently detect and identify cells, with the specific identification of CTCs as a future application through fluorescent labeling in mind. As proof of principle, the device has been shown to detect and characterize individual human Jurkat (T-lymphocyte) cells at a rate of 100 cells/minute. The device employs micro-scale flow focusing to isolate individual cells. The cells are detected using both light scattering and laser-induced fluorescence to evaluate cell size and surface functionality.

Polymere Nanopartikel sind ein vielversprechender Ansatz für die Diagnose und Therapie von Krankheiten. Sie ermöglichen den Einsatz von schwerlöslichen oder instabilen Wirkstoffen. Ein weiterer Vorteil ist die Möglichkeit das Targetings, durch gezielte Modifikationen des Nanopartikels wird der Wirkstoff zum Zielort transportiert und kann dort in der gewünschten Form freigesetzt werden; dadurch könnten bei erhöhter Wirksamkeit die Nebenwirkungen von Medikamenten reduziert werden. Ziel dieser Arbeit war die Untersuchung von physikalischen und biochemischen Eigenschaften von Nanopartikeln bestehend aus einem abbaustabilen Polystyren- Kern und einer biologisch abbaubaren Schale aus Polybutylcyanoacrylat. Es werden Methoden beschrieben, um die Größe, Struktur und den Abbau dieser Wirkstoffträger zu untersuchen. Die untersuchten Nanopartikel zeigen RAYLEIGH-Streuung, sowohl Größe als auch Abbau können durch Messung des Absorptionsspektrums bestimmt werden. Weiterhin konnten diese Eigenschaften mit Hilfe von dynamischer und statischer Lichtstreuung sowie Neutronenkleinwinkelstreuung untersucht werden. Bei letzterer Methode konnte gezeigt werden, dass die Schale größtenteils abgebaut werden kann, während der Kern intakt bleibt. In einem weiteren Teil der Arbeit wurde die Überwindung der Blut-Hirn-Schranke durch polymere Nanopartikel untersucht. Dabei wurde der fluoreszierende Thioflavine als Modellwirkstoffe eingesetzt. Das Durchdringen der Blut-Hirn-Schranke konnte nur mit Nanopartikeln erreicht werden, an deren Oberfläche ein Apolipoprotein E-Peptid gekoppelt war. Es konnte gezeigt werden, das die Nanopartikelschale im Gehirn abgebaut wird, der Wirkstoff freigesetzt wird und an Amyloid β, einem Marker der Alzheimer-Krankheit, bindet.

Liposomes are versatile biocompatible and biodegradable carriers for a variety of medical applications. As the first nanoparticles, they have been approved for pharmaceutical use so far, and many liposome-based preparations are in clinical trials. Classical methods of liposome preparation represent potential limitations in technology transfer from laboratory to industrial scale. New, microfluidic techniques overcome these limitations and offer new possibilities for controlled, continuous preparation of liposomal particles in a laboratory and industrial scale. An important element in the development of new nanoparticle systems is their complex characterization and purification. In addition to the established chromatographic techniques, the Field flow fractionation technique, in particular the Asymmetrical flow Field-flow fractionation, is described. This relatively new technique in conjunction with the MALS/DLS/DAD-UV/dRI online detectors enables the purification and characterization of complex samples. The main advantage of this technique lies in the possibility of separation under native conditions, which plays an important role in the separation of biopolymers in particular. Separation in the “empty” channel then eliminates sample degradation due to unwanted interactions at the stationary phase-sample interface. The theoretical part of this thesis describes the possibilities of preparation, modification, and characterization of liposomal nanoparticles. For this purpose, optical methods based on dynamic light scattering, multi-angle dynamic light scattering and nanoparticle tracking analysis techniques are described, as well as a non-optical method using "particle by the particle" analysis, tunable resistive pulse sensing method. A separate chapter of the theoretical part is dedicated to the technique Asymmetrical flow Field-flow fractionation in connection with the above-mentioned detectors. Important results associated with this work are summarized in the attached scientific paper, together with the result summaries and the author's contributions.

This thesis describes bioanalytical methods for drug determination in biological matrixes, with drugs in focus used against diseases largely affecting low-income countries. Solid-phase extraction is used for sample cleanup, and processed samples are analyzed by liquid chromatography. Developed bioanalytical methods are validated according to international guidelines. Eflornithine (DFMO) is a chiral drug, used for treating human African trypanosomiasis. A bioanalytical method for determination of DFMO enantiomers in plasma is presented. The enantiomers are detected by evaporative light-scattering detection. The method has been applied to determination of D-DFMO and L-DFMO in rats, after intravenous and oral administration of racemic DFMO. It is concluded that DFMO exhibits enantioselective absorption, with the more potent enantiomer L-DFMO being less favored. Sulfadoxine (SD) and sulfamethoxazole (SM) are sulfa-drugs used for malaria and pneumonia respectively. Two methods are described for simultaneous determination of SD and SM in capillary blood sampled on filter paper. The former method allows direct injection of extracts from dried blood spots (DBS), while for the latter method solid-phase extraction is added. Pre-analytical factors contributing to measurement uncertainty is also discussed, and it is concluded that it is of high importance that homogeneity in type of sampling paper and sampling volume is assured. Piperaquine (PQ) is an antimalarial, increasingly used in artemisinin combination therapy. A method for determination of piperaquine in DBS is presented. By using a monolithic LC column, a very short LC analysis of two minutes per sample is achieved. A method for simultaneous determination of three antiretroviral drugs i.e. lamivudine (3TC), zidovudine (AZT) and nevirapine (NVP), in DBS samples is described. The method is applied to drug determination in two subjects after receiving standard antiretroviral treatment. Conclusion is that the method is suitable for determination of 3TC and NVP, and to some extent for AZT.

Lab-on-Chip, the miniaturization of the chemical and analytical lab, is an endeavor that seems to come out of science fiction yet is slowly becoming a reality. It is a multidisciplinary field that combines different areas of science and engineering. Within these areas, microfluidics is a specialized field that deals with the behavior, control and manipulation of small volumes of fluids. Agglutination assays are rapid, single-step, low-cost immunoassays that use microspheres to detect a wide variety molecules and pathogens by using a specific antigen-antibody interaction. Agglutination assays are particularly suitable for the miniaturization and automation that two-phase microfluidics can offer, a combination that can help tackle the ever pressing need of high-throughput screening for blood banks, epidemiology, food banks diagnosis of infectious diseases. In this thesis, we present a two-phase microfluidic system capable of incubating and quantifying agglutination assays. The microfluidic channel is a simple fabrication solution, using laboratory tubing. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5-10 fold improvement over traditional agglutination assays. It has a user-friendly interface that that does not require droplet generators, in which a pipette is used to continuously insert assays on-demand, with no down-time in between experiments at 360 assays/h. System parameters are explored, using the streptavidin-biotin interaction as a model assay, with a minimum detection limit of 50 ng/mL using optical image analysis. We compare optical image analysis and light scattering as quantification methods, and demonstrate the first light scattering quantification of agglutination assays in a two-phase ow format. The application can be potentially applied to other biomarkers, which we demonstrate using C-reactive protein (CRP) assays. Using our system, we can take a commercially available CRP qualitative slide agglutination assay, and turn it into a quantitative High Sensitivity-CRP test, with a lower detection limit of 0.5 mg/L using light scattering. Agglutination assays are an incredibly versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as that presented in this thesis is a step towards being able to produce high throughput microfluidic solutions with widespread adoption.

One of the most studied subjects in Bionanotechnology is the application of Gold Nanoparticles (AuNPs). These have unique optical and chemical properties and interact with proteins and other biomolecules forming dynamic (Protein-Corona) layers at the surface. These protein coronas are responsible for increased in vivo biocompatibility, and can be studied by multiple techniques, tracking for disease-specific protein profiles. In this work, 15 nm AuNPs were synthesized by the Turkevich method, and 40 nm AuNPs were provided. Sample concentration and size were determined by UV-Vis spectroscopy, exploiting the Surface Plasmon Resonance (SPR) effect. Successful surface functionalization was performed with the alkanethiol 11-mercaptoundecanoic acid (MUA) or a pentapeptide (CALNN), maintaining a negative global net charge and increasing overall stability. Bionanoconjugation with Bovine Serum Albumin (BSA) and Fibrinogen (Fib), with molecular weights of 66 and 340 kDa respectively, was performed and characterized by Agarose Gel Electrophoresis (AGE). Electrophoretic mobility was determined using image and video analysis performed by the eReuss software. Adsorption affinity constant were determined using the conjugation curves obtained in the AGE results, fitted using the Langmuir Isotherm, and resulted in (1.5 ± 0.1) x 10-2 (AuNP-MUA) for BSA conjugation, and (51.2 ± 4.7) x 10-2 (AuNP-CALNN) and (34.3 ± 1.2) x 10-2 (AuNP-MUA) for Fib conjugation. Bioconjugation of AuNP-CALNN with BSA was inconclusive. Competitive scenarios of a protein mixture favored Fib adsorption over BSA. Fib conjugation of 40 nm AuNPs showed multiple adsorption constants of (3 ± 0.7) x 10-2 and (9.7 ± 2.2) x 10-4 respectively. The eReuss software proved to be a powerful tool to analyze image results from electrophoretic runs, and the video analysis feature gives way to an innovative way of analyzing these experiments and extract further information on the Protein Corona stability. Fergusson Plot analysis and Light scattering techniques (DLS, NTA and ELS) were performed to determine hydrodynamic sizes and Zeta-Potential of bionanoconjugated samples.
Uma das mais estudadas áreas em Bionanotecnologia é a aplicação de Nanopartículas de Ouro (AuNPs). Estas possuem propriedades óticas e químicas únicas e interagem com proteínas e outras biomoléculas, formando camadas dinâmicas a superfícies (Coroa Proteica). Estas coroas são responsáveis pelo aumento da biocompatibilidade in vivo, e podem ser estudadas com múltiplas técnicas, podendo identificar perfis de doença específicos. Neste trabalho, AuNPs de 15 nm foram sintetizadas pelo método de Turkevich, e AuNPs de 40 nm foram fornecidas. Concentração e tamanho das nanopartículas foram determinadas por espectroscopia UV-Vis, usando o efeito de Ressonância Plasmónica de Superfície (SPR). Funcionalização da superfície foi executada com adição de ácido 11-mercaptoundecanoico (MUA) e um penta-péptido (CALNN), mantendo a carga global negative e aumentando a estabilidade. Bioconjugação com Albumina (BSA) e Fibrinogénio (Fib) de soro bovino, com pesos moleculares de 66 e 340 kDa, respetivamente, foi executada e caracterizada por Eletroforese em Gel de Agarose (AGE). Mobilidade eletroforética foi determinada usando análise de imagem e vídeo com o programa eReuss. As constantes de afinidade de adsorção foram determinadas usando as curvas de conjugação pelos resultados de AGE, com a equação do modelo de adsorção de Langmuir, e resultou em (1.5 ± 0.1)x 10-2 (AuNP-MUA) para a conjugação com BSA, e (51.2 ± 4.7)x 10-2 (AuNP-CALNN) e (34.3 ± 1.2) x 10-2 (AuNP-MUA) para a conjugação com Fib. Bioconjugação de AuNP-CALNN com BSA foi inconclusiva. Cenários de competição numa mistura de proteínas favoreceu o Fib sobre a BSA. A conjugação de AuNPs de 40 nm mostrou múltiplas constantes de adsorção de (3 ± 0.7) x 10-2 e (9.7 ± 2.2) x 10-4 respetivamente. O programa eReuss provou ser uma poderosa ferramenta de análise de imagens das corridas eletroforéticas, e a componente de análise de vídeo sugere uma forma inovadora de analisar estas experiências e extrair informação adicional sobre a estabilidade da Coroa Proteica. A análise de Fergusson e técnicas de dispersão de luz (DLS, NTA e ELS) foram executadas para determinar o tamanho hidrodinâmico e o Potencial-Zeta de bionanoconjugados.

Noble metal nanoparticles constitute promising biosensors due to a high and tailored affinity to biomolecules such as proteins, forming protein coronas of distinct compositions on the surface. Gold nanoparticles (AuNP) are particularly interesting for a relatively easy, quick and inexpensive synthesis, low toxicity and ease of functionalization with bifunctional molecules, which typically have thiol groups bound to the AuNP surface and bio-friendly chemical groups at the opposite end, allowing for controlled protein adsorption. Functionalized AuNP can be used as probing agents for blood samples and health states determined by the protein corona composition, which is divided into a strongly bound innermost hard corona and a looser external soft corona. The objective of this work was to further understand the behaviour of plasma proteins integrating the corona. To attain these objectives, conjugates were prepared with ca. 15 and 40 nm diameter AuNP and two important plasma proteins: serum albumin and fibrinogen. AuNP synthesis was by a modified Turkevich method, with diameter and concentration determined by UV-Vis spectroscopy. AuNP were functionalized with 11-mercaptoundecanoic acid and conjugated with bovine or human serum albumin or fibrinogen. These single protein conjugates were evaluated for colloidal stability with ionic strength and pH variation through UV-Vis, protein conformational changes through circular dichroism (CD), hydrodynamic diameter changes upon centrifugation through dynamic light scattering (DLS) and electrophoretic mobility and concentration dependent conjugation efficiency were determined through agarose gel electrophoresis (AGE). Analysis of AGE profiles was by the open source electrophoresis gel image processing software eReuss. CD confirmed α-helix loss for conjugated serum albumins. DLS showed a hydrodynamic diameter decrease for centrifuged 42 nm AuNP conjugates, with high polydispersity indexes for 13 nm ones, suggesting aggregation. AGE revealed electrophoretic mobility decreases as the protein:AuNP ratio increases, data fitted to a Langmuir adsorption model. Serum albumin undergoes conformational alterations upon conjugation with AuNP. Centrifugation affects the protein corona, despite its tendency to aggregate AuNP. Overall, CD, DLS and AGE were demonstrated as useful techniques for the characterisation of the protein corona.

Upon arrival at the bloodstream, injected gold nanoparticles are covered with circulating plasma proteins, creating what is called a plasma corona. Its protein content is determined by the proteins’ affinity constants to the exposed surface of the nanoparticle. This work aims to propose an interaction mechanism between three plasma proteins and CALNN-functionalized nanoparticles via computational simulation and complementary experimental approach. Denaturing polyacrylamide gel electrophoresis determined the protein content of two human plasma samples, and helped in the characterization of the three most abundant blood proteins used in this study. Estimation of their electrostatic potential surfaces, silhouette areas, and diameters allowed the evaluation of the theoretical number of proteins forming a fully-covered nanoparticle. Seventeen transferrin molecules and eighteen albumin molecules with a side-on adsorption orientation were predicted to represent a monolayer adsorbome in a 20 nm gold nanoparticle. The dynamics of albumin adsorption to nanoparticles was studied through incubation-time assays on agarose gel electrophoresis, resulting in a stable protein corona starting from 7 h incubation time. The concentration ratio forming protein corona at the surface of nanoparticles was analysed through agarose gels electrophoretic mobility assays, revealing the formation of a full protein corona when a plateau in bionanoconjugates migration is achieved, resulting in protein coronas of [HSA]:[AuNP-CALNN] of 200:1 and 600:1 for [BPF]:[AuNP-CALNN] concentration ratios. Zeta-potential values were derived by relating agarose percentage with electrophoretic mobility of albumin bionanoconjugates, resulting in lower potential values for bionanoconjugates due to surface charge shielding of nanoparticles. Obtained ζ-potential values ranged from -26.05 up to -20.36 mV, forming colloid stable bionanoconjugates. Hydrodynamic radii of bionanoconjugates of albumin supported the formation of a monolayered and two-layered protein corona with increasing albumin:nanoparticle concentration ratios. Transferrin and fibrinogen showed increasing hydrodynamic radii with increasing protein:nanoparticle concentration ratios; in which fibrinogen bionanoconjugates showed fibrinogen wrapping around the nanoparticle. Electrostatic potential surfaces and protein-ligand docking using nanoparticle’s capping agent CALNN was performed in order to predict possible adsorption sites of human albumin and transferrin.

Cerebral blood flow (CBF) measures are central to the investigation of ischemic strokes, spreading depressions, functional and neuronal activation. Laser Speckle Contrast Imaging (LSCI) is an optical imaging technique that has been used to obtain CBF measures in vivo at high spatial and temporal resolutions, by quantifying the localized spatial blurring of backscattered coherent light induced by blood flow. Despite being widely used for biomedical applications, LSCI's critical limitations such as its tendency to underestimate large flow changes and its inability to accurately estimate CBF through a thinned skull have not been overcome. This dissertation presents a new Multi Exposure Speckle Imaging (MESI) technique that combines a new instrument and mathematical model to overcome these limitations. Additionally, in a pilot clinical study, an adapted neurosurgical microscope was used to obtain intra-operative LSCI images of CBF in humans. The MESI instrument accurately estimates experimental constants by imaging backscattered speckles over a wide range of the camera's exposure durations. The MESI mathematical model helps account for light that has scattered from both static and moving particles. In controlled flow experiments using tissue simulating phantoms, the MESI technique was found to estimate large changes in flow accurately and the estimates of flow changes were found to be unaffected by the presence of static particles in these phantoms. In an in vivo experiment in which the middle cerebral artery in mice was occluded to induce ~100% reduction in CBF, not only was the reduction in CBF accurately estimated by the MESI technique but these estimates of CBF changes were found to be unaffected by the presence of a thinned skull. The validity of statistical models used to derive the MESI mathematical model was confirmed using in vivo dynamic light scattering (DLS) measurements of CBF in mice. The MESI technique's potential to estimate absolute values of CBF in vivo was demonstrated by comparing CBF estimates obtained using the MESI technique to DLS measurements. The MESI technique's ability to measure CBF changes quantitatively through a thinned skull makes it particularly useful in chronic and long term studies leading to the development of better, more accurate stroke models.
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Polymere Nanopartikel sind ein vielversprechender Ansatz für die Diagnose und Therapie von Krankheiten. Sie ermöglichen den Einsatz von schwerlöslichen oder instabilen Wirkstoffen. Ein weiterer Vorteil ist die Möglichkeit das Targetings, durch gezielte Modifikationen des Nanopartikels wird der Wirkstoff zum Zielort transportiert und kann dort in der gewünschten Form freigesetzt werden; dadurch könnten bei erhöhter Wirksamkeit die Nebenwirkungen von Medikamenten reduziert werden. Ziel dieser Arbeit war die Untersuchung von physikalischen und biochemischen Eigenschaften von Nanopartikeln bestehend aus einem abbaustabilen Polystyren- Kern und einer biologisch abbaubaren Schale aus Polybutylcyanoacrylat. Es werden Methoden beschrieben, um die Größe, Struktur und den Abbau dieser Wirkstoffträger zu untersuchen. Die untersuchten Nanopartikel zeigen RAYLEIGH-Streuung, sowohl Größe als auch Abbau können durch Messung des Absorptionsspektrums bestimmt werden. Weiterhin konnten diese Eigenschaften mit Hilfe von dynamischer und statischer Lichtstreuung sowie Neutronenkleinwinkelstreuung untersucht werden. Bei letzterer Methode konnte gezeigt werden, dass die Schale größtenteils abgebaut werden kann, während der Kern intakt bleibt. In einem weiteren Teil der Arbeit wurde die Überwindung der Blut-Hirn-Schranke durch polymere Nanopartikel untersucht. Dabei wurde der fluoreszierende Thioflavine als Modellwirkstoffe eingesetzt. Das Durchdringen der Blut-Hirn-Schranke konnte nur mit Nanopartikeln erreicht werden, an deren Oberfläche ein Apolipoprotein E-Peptid gekoppelt war. Es konnte gezeigt werden, das die Nanopartikelschale im Gehirn abgebaut wird, der Wirkstoff freigesetzt wird und an Amyloid β, einem Marker der Alzheimer-Krankheit, bindet.

Σκοπός της διδακτορικής διατριβής είναι η ανάπτυξη κατάλληλων μεθόδων ψηφιακής επεξεργασίας εικόνας και αναγνώρισης προτύπων με τις οποίες θα προσδιορίζονται βιομετρικές και διαγνωστικές παράμετροι μέσω της αλληλεπίδρασης φωτονίων στο ορατό και υπέρυθρο φάσμα. Πιο συγκεκριμένα επιλύεται ένα αντίστροφο πρόβλημα σκέδασης ΗΜ ακτινοβολίας από ένα ανθρώπινο, υγιές και απαραμόρφωτο ερυθρό αιμοσφαίριο. Παρουσιάζονται μέθοδοι εκτίμησης και αναγνώρισης των γεωμετρικών χαρακτηριστικών απαραμόρφωτων υγιών ερυθρών αιμοσφαιρίων με χρήση εικόνων που προσομοιώνουν φαινόμενα σκέδασης ηλεκτρομαγνητικής ακτινοβολίας που διέρχεται από προσανατολισμένα ερυθρά αιμοσφαίρια. Η διαδικασία της ανάκτησης της πληροφορίας περιλαμβάνει, εξαγωγή χαρακτηριστικών με χρήση δισδιάστατων μετασχηματισμών, κανονικοποίηση των χαρακτηριστικών και την χρήση νευρωνικών δικτύων για την εκτίμηση των γεωμετρικών ιδιοτήτων του ερυθροκυττάρου. Παράλληλα σχεδιάστηκε και αξιολογήθηκε σύστημα αναγνώρισης των γεωμετρικών χαρακτηριστικών των ερυθρών αιμοσφαιρίων. Οι εικόνες σκέδασης δημιουργήθηκαν προσομοιώνοντας το πρόβλημα εμπρόσθιας σκέδασης ενός επίπεδου ηλεκτρομαγνητικού (ΗΜ) κύματος, χρησιμοποιώντας την μέθοδο των συνοριακών στοιχείων, λαμβάνοντας υπόψη τόσο την αξονοσυμμετρική γεωμετρία του ερυθροκυττάρου όσο και τις μη αξονοσυμμετρικές οριακές συνθήκες του προβλήματος. Η επίλυση του εν λόγω προβλήματος πραγματοποιήθηκε στα 632.8 nm και εν συνεχεία επεκτάθηκε σε 12 διακριτά ίσου βήματος μήκη κύματος από 432.8 nm έως 1032.8 nm. Επίσης, προτάθηκε μία νέα πειραματική διάταξη για την απόκτηση πολλαπλών εικόνων σκέδασης και την εκτίμηση των γεωμετρικών χαρακτηριστικών των ερυθρών αιμοσφαιρίων, αποτελούμενη από μία πολυχρωματική πηγή φωτός (Led) και πολλαπλά χρωματικά φίλτρα. Επίσης κατασκευάστηκε μέθοδος επίλυσης του σημαντικού προβλήματος εύρεσης της περιεκτικότητας του διαλύματος σε ερυθρά αιμοσφαίρια διαφορετικών μεγεθών στην περίπτωση απόκτησης πολλαπλών εικόνων σκέδασης από διαφορετικές φωτοδιόδους και πολλαπλά χρωματικά φίλτρα. Στα πειράματα αξιολόγησης της μεθόδου που προτείνεται με εικόνες προσομοίωσης δείχνεται ότι είναι ικανή η εύρεση της αναλογίας των ερυθρών αιμοσφαιρίων με πολύ μεγάλη ακρίβεια ακόμα και στη περίπτωση όπου στις εικόνες έχει προστεθεί λευκός κανονικός θόρυβος. Η βασική μεθοδολογία που παρουσιάζεται στην παρούσα δια-τριβή μπορεί να χρησιμοποιηθεί για την αναγνώριση παθολογικών αιμοσφαιρίων ή να χρησιμοποιηθεί στην αναγνώριση μικροσωματιδίων σε υγρά ή αέρια.
The aim of this PhD thesis is the development of digital image processing and pattern recognition methods to estimate biometric and diagnostic parameters using scattering phenomena in the visible and infrared spectrum. More concretely, several reverse scattering problems of EM radiation from a human, healthy and undistorted Red Blood Cell (RBC) is solved. Methods of estimation and recognition of geometrical characteristics of healthy and undistorted RBCs using simulating images are presented. The information retrieval process includes, features extraction using two-dimensional integral transforms, features normalization, and Neural Networks for estimation of three major RBC geometrical proper-ties. Using the same features set, a recognition system of the geometric characteristics of RBCs was developed and evaluated. The scattering images were created simulating the forward scattering problem of a plane electromagnetic wave using the Boundary Element Method, taking into account both axisymmetric geometry of the scatterer and the non-axisymmetric boundary conditions of the problem. Initially, the problem is solved at 632.8 nm and consequently the same problem was solved at 12 different wavelengths, from 432.8 to 1032.8 nm equally spaced. Also, a new device for acquisition of scattering images from RBCs-flow, consisting of a multi-color light source (Led) was proposed, for RBC size estimation and recognition. Finally, a system for the estimation of different RBCs concentration was developed when scattering images acquired using multiple scattering images acquired from multiple Leds and color filters. The system was evaluated using additive white regular noise.

Στην παρούσα διπλωματική εργασία θα γίνει μελέτη και εφαρμογή μεθόδων επίλυσης του προβλήματος αναγνώρισης γεωμετρικών χαρακτηριστικών ανθρώπινων ερυθρών αιμοσφαιρίων από προσομοιωμένες εικόνες σκέδασης ΗΜ ακτινοβολίας ενός He-Ne laser 632.8 μm. Στο πρώτο κεφάλαιο γίνεται μια εισαγωγή στις ιδιότητες και τα χαρακτηριστικά του ερυθροκυττάρου καθώς, επίσης, παρουσιάζονται διάφορες ανωμαλίες των ερυθροκυττάρων και οι μέχρι στιγμής χρησιμοποιούμενοι τρόποι ανίχνευσής των. Στο δεύτερο κεφάλαιο της εργασίας γίνεται μια εισαγωγή στις ιδιότητες της ΗΜ ακτινοβολίας, περιγράφεται το φαινόμενο της σκέδασης και παρουσιάζεται το ευθύ πρόβλημα σκέδασης ΗΜ ακτινοβολίας ανθρώπινων ερυθροκυττάρων. Το τρίτο κεφάλαιο αποτελείται από δύο μέρη. Στο πρώτο μέρος γίνεται εκτενής ανάλυση της θεωρίας των τεχνητών νευρωνικών δικτύων και περιγράφονται τα νευρωνικά δίκτυα ακτινικών συναρτήσεων RBF. Στη συνέχεια, αναφέρονται οι μέθοδοι εξαγωγής παραμέτρων και, πιο συγκεκριμένα, δίνεται το θεωρητικό και μαθηματικό υπόβαθρο των μεθόδων που χρησιμοποιήθηκαν οι οποίες είναι ο αλογόριθμος Singular Value Decomposition (SVD), o Angular Radial μετασχηματισμός (ART) και φίλτρα Gabor. Στο δεύτερο μέρος περιγράφεται η επίλυση του αντίστροφου προβλήματος σκέδασης. Παρουσιάζεται η μεθοδολογία της διαδικασίας επίλυσης όπου εφαρμόστηκαν ο αλογόριθμος συμπίεσης εικόνας SVD, o περιγραφέας σχήματος ART και ο περιγραφέας υφής με φίλτρα Gabor για την εύρεση των γεωμετρικών χαρακτηριστικών και νευρωνικό δίκτυο ακτινικών συναρτήσεων RBF για την ταξινόμηση των ερυθροκυττάρων. Στο τέταρτο και τελευταίο κεφάλαιο γίνεται δοκιμή και αξιολόγηση της μεθόδου και συνοψίζονται τα αποτελέσματα και τα συμπεράσματα που εξήχθησαν κατά τη διάρκεια της εκπόνησης αυτής της διπλωματικής.
In this thesis we study and implement methods of estimating the geometrical features of the human red blood cell from a set of simulated light scattering images produced by a He-Ne laser beam at 632.8 μm. Ιn first chapter an introduction to the properties and the characteristics of red blood cells are presented. Furthermore, we describe various abnormalities of erythrocytes and the until now used ways of detection. In second chapter the properties of electromagnetic radiation and the light scattering problem of EM radiation from human erythrocytes are presented. The third chapter consists of two parts. In first part we analyse the theory of neural networks and we describe the radial basis function neural network. Then, we describe the theoritical and mathematical background of the methods that we use for feature extraction which are Singular Value Decomposition (SVD), Angular Radial Transform and Gabor filters. In second part the solution of the inverse problem of light scattering is described. We present the methodology of the solution process in which we implement a Singular Value Decomposition approach, a shape descriptor with Angular Radial Transform and a homogenous texture descriptor which uses Gabor filters for the estimation of the geometrical characteristics and a RBF neural network for the classification of the erythrocytes. In the forth and last chapter the described methods are evaluated and we summarise the experimental results and conclusions that were extracted from this thesis.