Gold nanoparticles attain an intense focus in biomedical imaging applications due to their unique optical properties, facile conjugation with biomolecules, and biocompatibility. Although a considerable amount of work towards the development of gold nanoparticles has been completed, these promising contrast agents have not yet reached the clinic due to several challenges including efficient accumulation at the diseased site, sensitivity of detection in vivo, potential adverse effects, and clearance from the body. High signal-to-background ratio is required to enhance sensitivity of detection. Because near infrared (near-IR) light has the best tissue penetration, contrast agents designed to work in this range can significantly increase imaging sensitivity. Moreover, efficient targeting of the molecular biomarkers on diseased cells can decrease the required dosage, increase the site-specific accumulation, and enhance the imaging sensitivity. Molecular-specific contrast agents developed in this project use directional attachment of antibody molecules to the nanoparticle surface, enhancing the targeting efficacy. Additionally, cell-based delivery of diagnostic and therapeutic agents is gaining much interest due to the immune cells’ special access to the avascular, diseased regions. The contrast agents developed in this project enable detection of just a few cells per unit of imaging volume, enable multiplex imaging, and open up a possibility for tracking different cell populations with noninvasive photoacoustic and ultrasound imaging. Finally, the clearance of nanoparticles from the body dictates their clinical translation. The in vivo pharmacokinetics study along with the proposed in vitro model explored in this project will enable fast, reliable, and cost-efficient screening of promising agents and facilitate quick optimization of nanoparticles for their potential use in the clinic. / text

Cell migration is a fundamental biological process involved in tissue development, wound repair, and diseases such as cancer metastasis. It is a biomechanical process involving the adhesion of a cell to a substratum, usually an elastic extracellular matrix, as well as the physical contraction of the cell driven by intracellular actomyosin network. In the migration of cells as a group, known as collective migration, the cells are also physically linked to one another through cell-cell adhesions. How mechanical interactions with cell substratum and with neighboring cells regulate movements during collective migration, nevertheless, is poorly understood. To address this question, the effects of substrate stiffness on sheet migration of MCF10A epithelial cells were systematically analyzed. Speed, persistence, directionality and coordination of individual cells within the migrating sheet were all found to increase with substrate stiffening. Substrate stiffening also enhanced the propagation of coordinated movement from the sheet edge into the monolayer, which correlated with an upregulation of myosin-II activity in sheet edge cells. This mechano-response was dependent on cadherin-mediated cell-cell adhesions, which are required for the transmission of directional cue. Importantly, myosin-II contractility modulated cadherin- dependent cell-cell coordination, suggesting that contractile forces at cadherin adhesions regulate collective migration. To measure forces transmitted through cell-cell adhesions, a quantitative approach was developed in which cell-cell forces were deduced from cell-substrate traction forces, based on force balance principles and simple cell mechanics modeling. This method enabled the analysis of cell-cell mechanical interactions in small cell clusters of complex topology. The dynamic fluctuations of cell-cell forces over time revealed that force transmission between non-adjacent cells is typically limited, but is enhanced when the cell across which forces are being transmitted has reduced myosin-IIA or talin-1. This suggests that cells in a group may differentially regulate their levels of myosin-II contractility and cell-matrix mechanotransduction to promote longer-range force transmission during collective migration. Together, the results in this dissertation led to a working model of collective cell migration as regulated by cell-matrix mechanical properties and cell-cell mechanical interactions. This model, as well as the quantitative techniques developed here, will drive future studies on the mechanisms underlying collective migration.

This thesis focuses on cell tracking in image sequences acquired using a multimodal holographic microscope (MHM). The principles of holographic microscopy are described together with the application in cells acquisition. The main part of the thesis describes a complete approach for segmentation and tracking of single cells in acquired in long-term sequences. The approach is designed based on parametric active contour models with specific modifications to achieve reasonable precision and robustness. The implemented method is described in detail, including the evaluation and demonstration of results.

<p>In this dissertation, we document a few of our recent attempts in bridging the gap between the fast evolving deep learning research and the vast industry needs for dealing with computer vision challenges. More specifically, we developed novel deep-learning-based techniques for the following application-driven computer vision challenges: image super-resolution with quality restoration, motion estimation by optical flow, object detection for shape reconstruction, and object segmentation for motion tracking. Those four topics cover the computer vision hierarchy from the low level where digital images are processed to restore missing information for better human perception, to middle level where certain objects of interest are recognized and their motions are analyzed, finally to high level where the scene captured in the video footage will be interpreted for further analysis. In the process of building the whole-package of  ready-to-deploy solutions, we center our efforts on designing and training the most suitable convolutional neural networks for the particular computer vision problem at hand. Complementary procedures for data collection, data annotation,  post-processing of network outputs tailored for specific application needs, and deployment details will also be discussed where necessary. We hope our work demonstrates the applicability and versatility of convolutional neural networks for real-world computer vision tasks on a broad spectrum, from seeing better to understanding better.</p>

<p>This project is undergoing the patent application, so it is confidential and should not be disclosed. Further questions can be asked by contacting Dr. Jeroen Goos, whose contact information was shown in the supervisor section.</p>

In biology, many different kinds of microscopy are used to study cells. There are many different kinds of transmission microscopy, where light is passed through the cells, that can be used without staining or other treatments that can harm the cells. There is also fluorescence microscopy, where fluorescent proteins or dyes are placed in the cells or in parts of the cells, so that they emit light of a specific wavelength when they are illuminated with light of a different wavelength. Many fluorescence microscopes can take images on many different depths in a sample and thereby build a three-dimensional image of the sample. Fluorescence microscopy can also be used to study particles, for example viruses, inside cells. Modern microscopes often have digital cameras or other equipment to take images or record time-lapse video. When biologists perform experiments on cells, they often record image sequences or sequences of three-dimensional volumes to see how the cells behave when they are subjected to different drugs, culture substrates, or other external factors. Previously, the analysis of recorded data has often been done manually, but that is very time-consuming and the results often become subjective and hard to reproduce. Therefore there is a great need for technology for automated analysis of image sequences with cells and particles inside cells. Such technology is needed especially in biological research and drug development. But the technology could also be used clinically, for example to tailor a cancer treatment to an individual patient by evaluating different treatments on cells from a biopsy. This thesis presents algorithms to find cells and particles in images, and to calculate tracks that show how they have moved during an experiment. We have developed a complete system that can find and track cells in all commonly used imaging modalities. We selected and extended a number of existing segmentation algorithms, and thereby created a complete tool to find cell outlines. To link the segmented objects into tracks, we developed a new track linking algorithm. The algorithm adds tracks one by one using dynamic programming, and has many advantages over prior algorithms. Among other things, it is fast, it calculates tracks which are optimal for the entire image sequence, and it can handle situations where multiple cells have been segmented incorrectly as one object. To make it possible to use information about the velocities of the objects in the linking, we developed a method where the positions of the objects are preprocessed using a filter before the linking is performed. This is important for tracking of some particles inside cells and for tracking of cell nuclei in some embryos.       We have developed an open source software which contains all tools that are necessary to analyze image sequences with cells or particles. It has tools for segmentation and tracking of objects, optimization of settings, manual correction, and analysis of outlines and tracks. We developed the software together with biologists who used it in their research. The software has already been used for data analysis in a number of biology publications. Our system has also achieved outstanding performance in three international objective comparisons of systems for tracking of cells. / Inom biologi används många olika typer av mikroskopi för att studera celler. Det finns många typer av genomlysningsmikroskopi, där ljus passerar genom cellerna, som kan användas utan färgning eller andra åtgärder som riskerar att skada cellerna. Det finns också fluorescensmikroskopi där fluorescerande proteiner eller färger förs in i cellerna eller i delar av cellerna, så att de emitterar ljus av en viss våglängd då de belyses med ljus av en annan våglängd. Många fluorescensmikroskop kan ta bilder på flera olika djup i ett prov och på så sätt bygga upp en tre-dimensionell bild av provet. Fluorescensmikroskopi kan även användas för att studera partiklar, som exempelvis virus, inuti celler. Moderna mikroskop har ofta digitala kameror eller liknande utrustning för att ta bilder och spela in bildsekvenser. När biologer gör experiment på celler spelar de ofta in bildsekvenser eller sekvenser av tre-dimensionella volymer för att se hur cellerna beter sig när de utsätts för olika läkemedel, odlingssubstrat, eller andra yttre faktorer. Tidigare har analysen av inspelad data ofta gjorts manuellt, men detta är mycket tidskrävande och resultaten blir ofta subjektiva och svåra att reproducera. Därför finns det ett stort behov av teknik för automatiserad analys av bildsekvenser med celler och partiklar inuti celler. Sådan teknik behövs framförallt inom biologisk forskning och utveckling av läkemedel. Men tekniken skulle också kunna användas kliniskt, exempelvis för att skräddarsy en cancerbehandling till en enskild patient genom att utvärdera olika behandlingar på celler från en biopsi. I denna avhandling presenteras algoritmer för att hitta celler och partiklar i bilder, och för att beräkna trajektorier som visar hur de har förflyttat sig under ett experiment. Vi har utvecklat ett komplett system som kan hitta och följa celler i alla vanligt förekommande typer av mikroskopi. Vi valde ut och vidareutvecklade ett antal existerande segmenteringsalgoritmer, och skapade på så sätt ett heltäckande verktyg för att hitta cellkonturer. För att länka ihop de segmenterade objekten till trajektorier utvecklade vi en ny länkningsalgoritm. Algoritmen lägger till trajektorier en och en med hjälp av dynamisk programmering, och har många fördelar jämfört med tidigare algoritmer. Bland annat är den snabb, den beräknar trajektorier som är optimala över hela bildsekvensen, och den kan hantera fall då flera celler felaktigt segmenterats som ett objekt. För att kunna använda information om objektens hastighet vid länkningen utvecklade vi en metod där objektens positioner förbehandlas med hjälp av ett filter innan länkningen utförs. Detta är betydelsefullt för följning av vissa partiklar inuti celler och för följning av cellkärnor i vissa embryon. Vi har utvecklat en mjukvara med öppen källkod, som innehåller alla verktyg som krävs för att analysera bildsekvenser med celler eller partiklar. Den har verktyg för segmentering och följning av objekt, optimering av inställningar, manuell korrektion, och analys av konturer och trajektorier. Vi utvecklade mjukvaran i samarbete med biologer som använde den i sin forskning. Mjukvaran har redan använts för dataanalys i ett antal biologiska publikationer. Vårt system har även uppnått enastående resultat i tre internationella objektiva jämförelser av system för följning av celler. / <p>QC 20161125</p>

As research and clinical use of stem cell therapies progresses, it is becoming more evident that being able to visualize the stem cell transplant in vivo is of great benefit to the researcher or clinician. As a result, researchers are working to address this need. Our lab is collaborating to develop novel, multimodal nanomaterials, one with a core of mesoporous silica, and the other with a core of gadolinium oxide. Varying modifications have been made as needs arose. Human mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates and confirmed to be positive for STRO-1, a common MSC marker. These cells were labeled with 125 μg/mL of varying nanoparticle types: gadolinium oxide, doped with 0.5%, 5%, or 10% europium for magnetic resonance imaging (MRI) and luminescence microscopy, and mesoporous silica nanoparticles (MSN), loaded with fluorescein for fluorescent microscopy and capped with either iron oxide or gold for MRI and computed tomography (CT), respectively. We studied the kinetics of MSN uptake by MSCs for 10 days using fluorescent microscopy. In ex vivo studies, we used the 4.7 Tesla Varian® small animal MRI scanner to detect 5*10⁴ cells labeled with ferrite-capped MSN particles and injected into the brain, lung, and heart of a perfusion-fixed mouse. Micro-CT was used to detect 1.7*10⁶ cells labeled with gold-capped MSN and delivered to the lungs via the trachea in a perfusion-fixed mouse. The results of this research are preliminary to in vivo testing using animal models as a proof-of-concept for these potentially marketable particles.

In the presence of a solid-liquid or liquid-air interface, bacteria can choose between a planktonic and a sessile lifestyle. Depending on environmental conditions, cells swimming in close proximity to the interface can irreversibly attach to the surface and grow into three-dimensional aggregates where the majority of cells is sessile and embedded in an extracellular polymer matrix (biofilm). We used microfluidic tools and time lapse microscopy to perform experiments with the polarly flagellated soil bacterium Pseudomonas putida (P. putida), a bacterial species that is able to form biofilms. We analyzed individual trajectories of swimming cells, both in the bulk fluid and in close proximity to a glass-liquid interface. Additionally, surface related growth during the early phase of biofilm formation was investigated. In the bulk fluid, P.putida shows a typical bacterial swimming pattern of alternating periods of persistent displacement along a line (runs) and fast reorientation events (turns) and cells swim with an average speed around 24 micrometer per second. We found that the distribution of turning angles is bimodal with a dominating peak around 180 degrees. In approximately six out of ten turning events, the cell reverses its swimming direction. In addition, our analysis revealed that upon a reversal, the cell systematically changes its swimming speed by a factor of two on average. Based on the experimentally observed values of mean runtime and rotational diffusion, we presented a model to describe the spreading of a population of cells by a run-reverse random walker with alternating speeds. We successfully recover the mean square displacement and, by an extended version of the model, also the negative dip in the directional autocorrelation function as observed in the experiments. The analytical solution of the model demonstrates that alternating speeds enhance a cells ability to explore its environment as compared to a bacterium moving at a constant intermediate speed. As compared to the bulk fluid, for cells swimming near a solid boundary we observed an increase in swimming speed at distances below d= 5 micrometer and an increase in average angular velocity at distances below d= 4 micrometer. While the average speed was maximal with an increase around 15% at a distance of d= 3 micrometer, the angular velocity was highest in closest proximity to the boundary at d=1 micrometer with an increase around 90% as compared to the bulk fluid. To investigate the swimming behavior in a confinement between two solid boundaries, we developed an experimental setup to acquire three-dimensional trajectories using a piezo driven objective mount coupled to a high speed camera. Results on speed and angular velocity were consistent with motility statistics in the presence of a single boundary. Additionally, an analysis of the probability density revealed that a majority of cells accumulated near the upper and lower boundaries of the microchannel. The increase in angular velocity is consistent with previous studies, where bacteria near a solid boundary were shown to swim on circular trajectories, an effect which can be attributed to a wall induced torque. The increase in speed at a distance of several times the size of the cell body, however, cannot be explained by existing theories which either consider the drag increase on cell body and flagellum near a boundary (resistive force theory) or model the swimming microorganism by a multipole expansion to account for the flow field interaction between cell and boundary. An accumulation of swimming bacteria near solid boundaries has been observed in similar experiments. Our results confirm that collisions with the surface play an important role and hydrodynamic interactions alone cannot explain the steady-state accumulation of cells near the channel walls. Furthermore, we monitored the number growth of cells in the microchannel under medium rich conditions. We observed that, after a lag time, initially isolated cells at the surface started to grow by division into colonies of increasing size, while coexisting with a comparable smaller number of swimming cells. After 5:50 hours, we observed a sudden jump in the number of swimming cells, which was accompanied by a breakup of bigger clusters on the surface. After approximately 30 minutes where planktonic cells dominated in the microchannel, individual swimming cells reattached to the surface. We interpret this process as an emigration and recolonization event. A number of complementary experiments were performed to investigate the influence of collective effects or a depletion of the growth medium on the transition. Similar to earlier observations on another bacterium from the same family we found that the release of cells to the swimming phase is most likely the result of an individual adaption process, where syntheses of proteins for flagellar motility are upregulated after a number of division cycles at the surface. / Bakterien sind einzellige Mikroorganismen, die sich in flüssigem Medium mit Hilfe von rotierenden Flagellen, länglichen Fasern aus Proteinen, schwimmend fortbewegen. In Gegenwart einer Grenzfläche und unter günstigen Umweltbedingungen siedeln sich Bakterien an der Oberfläche an und gehen in eine sesshafte Wachstumsphase über. Die Wachstumsphase an der Oberfläche ist gekennzeichnet durch das Absondern von klebrigen, nährstoffreichen extrazellulären Substanzen, welche die Verbindung der Bakterien untereinander und mit der Oberfläche verstärken. Die entstehenden Aggregate aus extrazellulärer Matrix und Bakterien werden als Biofilm bezeichnet. In der vorliegenden Arbeit untersuchten wir ein Bodenbakterium, Pseudomonas putida (P. putida), welches in wässriger Umgebung an festen Oberflächen Biofilme ausbildet. Wir benutzten photolithographisch hergestellte Mikrokanäle und Hochgeschwindigkeits-Videomikroskopie um die Bewegung schwimmender Zellen in verschiedenen Abständen zu einer Glasoberfläche aufzunehmen. Zusätzlich wurden Daten über das parallel stattfindende Wachstum der sesshaften Zellen an der Oberfläche aufgezeichnet. Die Analyse von Trajektorien frei schwimmender Zellen zeigte, dass sich Liniensegmente, entlang derer sich die Zellen in eine konstante Richtung bewegen, mit scharfen Kehrtwendungen mit einem Winkel von 180 Grad abwechseln. Dabei änderte sich die Schwimmgeschwindigket von einem zum nächsten Segment im Mittel um einen Faktor von 2. Unsere experimentellen Daten waren die Grundlage für ein mathematisches Modell zur Beschreibung der Zellbewegung mit alternierender Geschwindigkeit. Die analytische Lösung des Modells zeigt elegant, dass eine Population von Bakterien, welche zwischen zwei Geschwindigkeiten wechseln, signifikant schneller expandiert als eine Referenzpopulation mit Bakterien konstanter Schwimmgeschwindkeit. Im Vergleich zu frei schwimmenden Bakterien beobachteten wir in der Nähe der Oberfläche eine um 15% erhöhte Schwimmgeschwindigkeit der Zellen und eine um 90 % erhöhte Winkel-geschwindigkeit. Außerdem wurde eine signifikant höhere Zelldichte in der Nähe der Grenzfläche gemessen. Während sich der Anstieg in der Winkelgeschwindigkeit durch ein Drehmoment erklären lässt, welches in Oberflächennähe auf den rotierenden Zellkörper und die rotierenden Flagellen wirkt, kann die Beschleunigung und Akkumulation der Zellen bei dem beobachteten Abstand nicht durch existierende Theorien erklärt werden. Unsere Ergebnisse lassen vermuten, dass neben hydrodynamischen Effekten auch Kollisionen mit der Oberfläche eine wichtige Rolle spielen und sich die Rotationsgeschwindigkeit der Flagellenmotoren in der Nähe einer festen Oberfläche grundsätzlich verändert. Unsere Experimente zum Zellwachstum an Oberflächen zeigten, dass sich etwa sechs Stunden nach Beginn des Experiments größere Kolonien an der Kanaloberfläche auflösen und Zellen für ca. 30 Minuten zurück in die schwimmende Phase wechseln. Ergebnisse von mehreren Vergleichsexperimenten deuten darauf hin, dass dieser Übergang nach einer festen Anzahl von Zellteilungen an der Oberfläche erfolgt und nicht durch den Verbrauch des Wachstumsmediums bedingt wird.

The local microenvironment of hematopoietic stem cells (HSCs) in the bone marrow -referred to as stem cell niche- is thought to regulate the balance of stem cell maintenance and differentiation by a complex interplay of extrinsic signals including spatial constraints, extracellular matrix (ECM) components and cell-cell interactions. To dissect the role of niche ECM components, a set of well-defined matrix biomolecular coatings including fibronectin, laminin, collagen IV, tropocollagen I, heparin, heparan sulphate, hyaluronic acid and co-fibrils of collagen I with heparin or hyaluronic acid were prepared and analyzed with respect to adhesive interactions of human CD133+ HSCs in vitro. ECM molecule dependent adhesion areas as well as fractions of adherent HSCs were assessed by reflection interference contrast microscopy and differential interference contrast microscopy. HSCs, so far mostly classified as suspension cells, exhibited intense adhesive interactions with fibronectin, laminin, collagen IV, heparin, heparan sulphate, and collagen I based co-fibrils. An integrin mediated adhesion on fibronectin and a L-selectin mediated adhesion on heparin pointed to specific interactions based on different adhesion mechanisms. As a consequence of HSC adhesion to molecules of the vascular and the endosteal regions, both regions were confirmed as possible stem cell niches and adhesive signals were suggested as potential regulators of stem cell fate. Furthermore, the impact of a spatially organized ECM on the HSC behavior was analyzed by single cell tracking. These studies required the development of engineered three-dimensional, ECM coated microcavities with the option for single cell tracking. A semi-automated cell-tracking tool was established to accelerate data access from time-lapse image sequences. From this analysis it was possible to reveal the genealogy, localization, morphology and migration of single HSCs over a time period of 4 days. A decreased cycling frequency was observed depending on the HSC localization in the spatially constraining microcavities. Besides the revealed impact of spatial constraints on HSC fate, the newly engineered ECM-coated microcavity setup and the semi-automated cell tracking tool provide new options to study the cell fate in engineered microenvironments at single cell level for other cell types ex vivo. / Die lokale Mikroumgebung von Blutstammzellen (BSZ) im Knochenmark, bezeichnet als Stammzellnische, reguliert das Gleichgewicht von Stammzellerhaltung und -differenzierung durch ein komplexes Zusammenspiel von extrinsischen Signalen wie räumliche Beschränkungen, Komponenten der extrazellulären Matrix (EZM) und Zell-Zell Wechselwirkungen. Um die Rolle der EZM-Komponenten zu analysieren, wurden definierte Beschichtungen von Fibronektin, Laminin, Kollagen IV, monomerem Kollagen I, Heparin, Heparan Sulphat, Hyaluronsäure und Co-Fibrillen aus Kollagen I und Heparin oder Hyaluronsäure hergestellt und in vitro bezüglich der adhäsiven Wechselwirkungen von humanen CD133+ BSZ untersucht. Die Adhäsionsflächen und der Anteil adhärenter Zellen wurden in Abhängigkeit von der EZM-Beschichtung mittels Reflexions- Interferenz-Kontrast-Mikroskopie und Differentieller Interferenz Kontrast Mikroskopie bestimmt. BSZ, bisher als Suspensionszellen definiert, zeigten intensive adhäsive Wechselwirkungen mit Fibronektin, Laminin, Kollagen IV, Heparin, Heparan Sulphat und den Co-Fibrillen. Eine Integrin abhängige Adhäsion auf Fibronektin und eine L-Selektin abhängige Adhäsion auf Heparin, wiesen auf spezifische Wechselwirkungen hin, die auf unterschiedlichen Mechanismen basieren. Aufgrund der Adhäsion von BSZ sowohl zu Molekülen der vaskulären als auch der endostealen Knochenmarkregion, wurden beide Bereiche als mögliche Stammzellnische bestätigt. Adhäsive Signale sind potentielle Regulatoren der Stammzellentwicklung. Im Weiteren wurde der Einfluss einer räumlich beschränkenden EZM auf das Verhalten der BSZ durch Einzelzellverfolgung untersucht. Diese Studien erforderten die Entwicklung von dreidimensionalen EZM-beschichteten Mikrokavitäten, die das Verfolgen einzelner Zellen ermöglichten. Es wurde ein halbautomatischer Algorithmus für die Zellverfolgung etabliert, um die Datengenerierung von den Zeitreihenaufnahmen zu beschleunigen. Die Analysen ermöglichten Aussagen über die Genealogie, Lokalisierung, Morphologie und Migration einzelner BSZ während einer Analysenzeit von 4 Tagen. Eine verringerte Zellteilungsaktivität wurde in Abhängigkeit von der BSZ Lokalisierung innerhalb der räumlich einschränkenden Mikrokavitäten festgestellt. Neben diesen Erkenntnissen bieten die entwickelten Mikrokavitäten und die etablierte Einzelzellverfolgung neue Möglichkeiten auch andere Zelltypen auf Einzelzellniveau ex vivo zu untersuchen.

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