Cette thèse est centrée sur la synthèse, la caractérisation, la fonctionnalisation et les études in vitro et in vivo de différents types de nanoparticules (NPs) de très petite taille (NPs de silicium ; « carbon dots » ; NPs de cobalt couvertes de silice) qui présentent une luminescence ou des propriétés magnétiques et qui peuvent être fonctionnalisées par des liaisons covalentes. Tous les systèmes étudiés sont très intéressants pour l'imagerie multimodale in vivo, ce qui nécessite l'ancrage stable de fonctionnalités multiples sur une seule plate-forme nanométrique. Dans cette perspective, les NPs ont été fonctionnalisées avec des groupements amine et, puis, couplées avec des colorants ou des marqueurs radioactifs afin d'effectuer, in vivo, de l’imagerie optique ou de tomographie d'émission des positrons. Lorsqu’elles sont étudiées in vitro, les NPs ont présenté une cytotoxicité très faible. Les expériences in vivo ont donné des résultats très prometteurs puisque les NPs ont été excrétées par le corps en très peu de temps, principalement par un mécanisme de clairance rénale, avec une faible accumulation dans les organes. / This thesis focuses on the synthesis, characterization, functionalization and in vitro and in vivo investigation of different kinds of ultrasmall nanoparticles (silicon nanoparticles: Si NPs; carbon dots: CQDs; silica-coated cobalt NPs: Co@SiO2 NPs) which exhibit luminescence or magnetic properties and can be functionalized through covalent bonds. All the systems studied are very attractive for in vivo multimodal imaging, which requires the stable anchoring of multiple functionalities on a single nano-sized platform. In this perspective, nanoparticles were functionalized with amine-moieties and, subsequently, coupled with dyes or radiolabels in order to perform in vivo optical or positron emission tomography imaging. When tested in vitro, ultrasmall nanoparticles showed very low cytotoxicity. In vivo experiments gave very promising results since nanoparticles were excreted from the body in short times, mainly through a renal clearance mechanism, with low accumulation in organs.

Recent evidence suggests that cardiac mechanics (e.g. cardiac strains) are better measures of heart function compared to common clinical metrics like ejection fraction. However, commonly-used parameters of cardiac mechanics remain limited to just a few measurements averaged over the whole left ventricle. We hypothesized that recent advances in cardiac magnetic resonance imaging (MRI) could be extended to provide measures of cardiac mechanics throughout the left and right ventricles (LV and RV, respectively). Displacement Encoding with Stimulated Echoes (DENSE) is a cardiac MRI technique that has been validated for measuring LV mechanics at a magnetic field strength of 1.5 T but not at higher field strengths such as 3.0 T. However, it is desirable to perform DENSE at 3.0 T, which would yield a better signal to noise ratio for imaging the thin RV wall. Results in Chapter 2 support the hypothesis that DENSE has similar accuracy at 1.5 and 3.0 T. Compared to standard, clinical cardiac MRI, DENSE requires more expertise to perform and is not as widely used. If accurate mechanics could be measured from standard MRI, the need for DENSE would be reduced. However, results from Chapter 3 support the hypothesis that measured cardiac mechanics from standard MRI do not agree with, and thus cannot be used in place of, measurements from DENSE. Imaging the thin RV wall with its complex contraction pattern requires both three-dimensional (3D) measures of myocardial motion and higher resolution imaging. Results from Chapter 4 support the hypothesis that a lower displacement-encoding frequency can be used to allow for easier processing of 3D DENSE images. Results from Chapter 5 support the hypothesis that images with higher resolution (decreased blurring) can be achieved by using more spiral interleaves during the DENSE image acquisition. Finally, processing DENSE images to yield measures of cardiac mechanics in the LV is relatively simple due to the LV’s mostly cylindrical geometry. Results from Chapter 6 support the hypothesis that a local coordinate system can be adapted to the geometry of the RV to quantify mechanics in an equivalent manner as the LV. In summary, cardiac mechanics can now be quantified throughout the left and right ventricles using DENSE cardiac MRI.

Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further. Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016. Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems. In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact. / Avbildning av biologiska prover har i flera hundra år varit ett sätt för forskare att undersöka biologiska system. Med utvecklingen av immunofluoresens inmärkn-ing och fluoresens-mikroskopi förbättrades de viktigaste aspekterna av mikroskopi,kontrast och specificitet. Sedan 1941 har vi sett kontinuerligt mer mångsidigt och frekvent användning av fluorosense-mikroskopi i biologisk forskning. Jon-mikroskopi har länge varit en metod att studera signalering i cell-system. Genom användning av fluorosenta jon-sensorer går det att mäta variationer avjon koncentrationer i levande celler som resultat av yttre påverkan. Genom att använda Ca2+ mikroskopi har jag visat att det finns en omvänd koppling mellan kalcium-kanaler i plasma-membran och angiotensin II typ 1 receptorn (ett proteininvolverat i blodtrycksreglering). Detta har direkta implikationer för behandlingav högt blodtryck, en av de mer vanliga sjukdomarna i västvärlden idag med överen miljard drabbade patienter i världen 2016. Efter detta projekt vidgades mitt fokus till att inkludera superupplösnings-mikroskopi. Denna avhandling inkluderar ett arbete fokuserat på tolkningen av superupplösnings-mikroskopi data från neuronal Na+, K+ - ATPase α3, en jon-pump som återställer cellernas jonbalans i samband med cell signalering. Mikroskopi-datan korreleras mot elektrofysiologi experiment och modeller för att illustrera möjliga artefakter i superupplösnings-mikroskopi som måste tas i beaktande i samband med tolkning av data. Jag fortsatte med att utveckla mjukvara för analys av data från singel-molekyl-lokalisations-mikroskopi där fokuset för mjukvaran framförallt varit på användarvänligheten. Detta då jag hoppas att den kommer vara användbar för ett bredare forskingsfält. Mjukvaran användes även i ett separat projekt för att identifiera överuttrycks-artefakter i transfekterade celler. I det avslutande arbetet använder jag superupplösnings-mikroskopi för att karakterisera de tidiga stegen i mitokondriell apoptos. Jag identifierar när och var i cellen de olika proteinerna involverade i apoptos signaleringen är aktiverade och interagerar. / <p>QC 20171003</p>

Optical coherence tomography (OCT) and Photoacoustic microscopy (PAM) are two noninvasive, high-resolution, three-dimensional, biomedical imaging modalities based on different contrast mechanisms. OCT detects the light backscattered from a biological sample either in the time or spectral domain using an interferometer to form an image. PAM is sensitive to optical absorption by detecting the light-induced acoustic waves to form an image. Due to their complementary contrast mechanisms, OCT and PAM are suitable for being combined to achieve multimodal imaging. In this dissertation, an optical coherence photoacoustic microscopy (OC-PAM) system was developed for in vivo multimodal retinal imaging with a pulsed broadband NIR light source. To test the capabilities of the system on multimodal ophthalmic imaging, the retina of pigmented rats was imaged. The OCT images showed the retinal structures with quality similar to conventional OCT, while the PAM images revealed the distribution of melanin in the retina since the NIR PAM signals are generated mainly from melanin in the posterior segment of the eye. By using the pulsed broadband light source, the OCT image quality highly depends on the pulse-to-pulse stability of the light source without averaging. In addition, laser safety is always a concern for in vivo applications, especially for eye imaging with a pulsed light source. Therefore, a continuous wave (CW) light source is desired for OC-PAM applications. An OC-PAM system using an intensity-modulated CW superluminescent diode was then developed. The system was tested for multimodal imaging the vasculature of a mouse ear in vivo by using Gold Nanorods (GNRs) as contrast agent for PAM, as well as excised porcine eyes ex vivo. Since the quantitative information of the optical properties extracted from the proposed NIR OC-PAM system is potentially able to provide a unique technique to evaluate the existence of melanin and lipofuscin specifically, a phantom study has been conducted and the relationship between image intensity of OCT and PAM was interpreted to represent the relationship between the optical scattering property and optical absorption property. It will be strong evidence for practical application of the proposed NIR OC-PAM system.

For most of the patients with brain tumors and/or epilepsy, surgical resection of brain lesions, when applicable, remains one of the optimal treatment options. The success of the surgery hinges on accurate demarcation of neoplastic and epileptogenic brain tissue. The primary goal of this PhD dissertation is to demonstrate the feasibility of using various optical techniques in conjunction with sophisticated signal processing algorithms to differentiate brain tumor and epileptogenic cortex from normal brain tissue intraoperatively. In this dissertation, a new tissue differentiation algorithm was developed to detect brain tumors in vivo using a probe-based diffuse reflectance spectroscopy system. The system as well as the algorithm were validated experimentally on 20 pediatric patients undergoing brain tumor surgery at Nicklaus Children’s Hospital. Based on the three indicative parameters, which reflect hemodynamic and structural characteristics, the new algorithm was able to differentiate brain tumors from the normal brain with a very high accuracy. The main drawbacks of the probe-based system were its high susceptibility to artifacts induced by hand motion and its interference to the surgical procedure. Therefore, a new optical measurement scheme and its companion spectral interpretation algorithm were devised. The new measurement scheme was evaluated both theoretically with Monte Carlo simulation and experimentally using optical phantoms, which confirms the system is capable of consistently acquiring total diffuse reflectance spectra and accurately converting them to the ratio of reduced scattering coefficient to absorption coefficient (µs’(λ)/µa(λ)). The spectral interpretation algorithm for µs’(λ)/µa(λ) was also validated based on Monte Carlo simulation. In addition, it has been demonstrated that the new measurement scheme and the spectral interpretation algorithm together are capable of detecting significant hemodynamic and scattering variations from the Wistar rats’ somatosensory cortex under forepaw stimulation. Finally, the feasibility of using dynamic intrinsic optical imaging to distinguish epileptogenic and normal cortex was validated in an in vivo study involving 11 pediatric patients with intractable epilepsy. Novel data analysis methods were devised and applied to the data from the study; identification of the epileptogenic cortex was achieved with a high accuracy.

This thesis presents a novel ZnO-hydrogel based fluorescent colloidal semiconductor nanomaterial system for potential bio-medical applications such as bio-imaging, cancer detection and therapy. The preparation of ZnO nanoparticles and their surface modification to make a biocompatible material with enhanced optical properties is discussed. High quality ZnO nanoparticles with UV band edge emission are prepared using gas evaporation method. Semiconductor materials including ZnO are insoluble in water. Since biological applications require water soluble nanomaterials, ZnO nanoparticles are first dispersed in water by ball milling method, and their aqueous stability and fluorescence properties are enhanced by incorporating them in bio-compatible poly N-isopropylacrylamide (PNIPAM) based hydrogel polymer matrix. The optical properties of ZnO-hydrogel colloidal dispersion versus ZnO-Water dispersion were analyzed. The optical characterization using photoluminescence spectroscopy indicates approximately 10 times enhancement of fluorescence in ZnO-hydrogel colloidal system compared to ZnO-water system. Ultrafast time resolved measurement demonstrates dominant exciton recombination process in ZnO-hydrogel system compared to ZnO-water system, confirming the surface modification of ZnO nanoparticles by hydrogel polymer matrix. The surface modification of ZnO nanoparticles by hydrogel induce more scattering centers per unit area of cross-section, and hence increase the luminescence from the ZnO-gel samples due to multiple path excitations. Furthermore, surface modification of ZnO by hydrogel increases the radiative efficiency of this hybrid colloidal material system thereby contributing to enhanced emission.

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (pIn vivomouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p

The mapping of the refractive index of cells has been extensively studied since 1950s. This optical parameter constitutes a key biophysical property strongly correlated to fundamental cell parameters such, e.g., intracellular mass distribution and protein concentration. Experimental studies evidence that the cell refractive index (Refractive Index) provides critical insights to understand diverse cellular structures and interpret pathological states, including diverse stages of diseases. However, measuring the refractive indices of biological specimens satisfying clinical requirements is currently challenging, since there is a lack of spectral signatures of sub-cellular components in the visible range due to their transparent nature. Designing methods capable of extracting visible fingerprints of cellular components remains attracting large research interests. In this work, I have contributed to this project by fabricating and characterizing a black nanostructured membrane that dynamically interacts with cancerous cells and furnishes label-free structural color generation by exploiting the inherent contrast mechanisms of them. Thus, adequately meeting morphology differentiation to assist in biomedical research. I have tested the system with HCT116 colorectal cancer cells. In addition, this special membrane allows refractive index recovery and cell thickness mapping with commonly available bright-field microscopy equipment. Therefore, it is of considerable clinical importance to allow the generation of qualitative information about cell morphology to add in medicine and biophysics research.

Atopic Dermatitis (AD), a chronic inflammatory skin disease causing lesions, often causes decreased quality of life (Kapur, 2018). Segmentation, a method of illustrating the difference between lesioned and non-lesioned areas of interest (AOIs) has been the primary method for which AD has been studied (Ranteke & Jain, 2013). Manual segmentation is prone to subjectivity (Ning et al., 2014) and automatic segmentation, while reliable and efficient, poses challenges such as light reflections and color variations (Lu et al., 2013). Yet, AD can be classified from color and texture (Hanifin et al., 2001; Nisar et al., 2013), as well as through machine learning methods. The purpose of this study was to determine the optimal method for segmentation of images of atopic dermatitis on subject arms in a novel and standardized photography lightbox (Lightbox) and of images of subjects' self-acquired at-home photos. The goals of this study were to determine the accuracy and reliability of photo acquisition of arms of subjects with AD in a novel standardized photography lightbox, compared to photo acquisition by subjects at home, and determine the accuracy and reliability of automated segmentation of AD lesions with combined color-based segmentation and the U-Net CNN.

Ces dernières années, la théorie mathématique de l'échantillonnage compressé (CS) a émergé en tant que nouvel outil en traitement d'images, permettant notamment de dépasser certaines limites établies par la théorie de l'échantillonnage de Nyquist. En particulier, la théorie du CS établit qu'un signal (une image, une séquence vidéo, etc.) peut être reconstruit à partir d'un faible nombre de mesures linéaires non-adaptatives et aléatoires, pourvu qu'il présente une structure parcimonieuse. Dans la mesure où cette hypothèse se vérifie pour une large classe d'images naturelles, plusieurs applications d'imagerie ont d'ores-et-déjà bénéficié à des titres divers des résultats issus de cette théorie. Le but du travail doctoral présent est d'étudier comment la théorie du CS - et plus généralement les idées et méthodes en relation avec les problèmes de reconstruction de signaux parcimonieux - peuvent être utilisés pour concevoir des dispositifs d'acquisition optiques à haute-résolution spatiale et temporelle pour des applications en imagerie biologique. Nous étudions tout d'abord quelques questions pratiques liées à l'étape de reconstruction nécessairement associée aux systèmes d'acquisition exploitant le CS, ainsi qu'à la sélection des paramètres d'échantillonnage. Nous examinons ensuite comment le CS peut être utilisé dans le cadre d'applications d'échantillonnage de signaux vidéo. Enfin, avec dans l'idée l'utilisation dans des problèmes de débruitage de méthodes inspirées du CS, nous abordons la question de l'estimation d'erreur dans les problèmes de débruitage d'images acquises en conditions de faible luminosité, notamment dans le cadre d'applications de microscopie. / In the past few years, the mathematical theory of compressed sensing (CS) has emerged as a new tool in the image processing field, leading to some progress in surpassing the limits stated by the Nyquist sampling theory. In particular, the CS theory establishes that a signal (image, video, etc.) can be reconstructed from a relatively small subset of non-adaptive linear random measurements, assuming that it presents a sparse structure. As this hypothesis actually holds for a large number of natural images, several imaging applications have already benefited from this theory in various aspects. The goal of the present PhD work is to investigate how the CS theory - and more generally the ideas and methods developed in relation with sparse signal reconstruction problematics - can be used to design efficient optical sensing devices with high spatial and temporal resolution for biological imaging applications. We first investigate some practical issues related to the post-processing stage required by CS acquisition schemes, and to the selection of sampling parameters. We then examine how CS can benefit to video sampling applications. Finally, with the application of CS methods for denoising tasks in mind, we focus on the error estimation issue in image denoising problems for low-light microscopy applications.