Application of the nonlinear optical microscopy (NLOM) for investigation of biological samples has, to date, primarily focused upon the qualitative analysis of images. The general consensus is that the nonlinear optical (NLO) techniques provide enough bio- chemical information when compared to, for example, visible light microscopy. Herein, it is presented a detailed study where a set of tools for quantitative extraction of infor- mation from NLO images were developed and tested for the analysis of complex tissue assemblies. Two-photon excited autofluorescence (TPEF), second-harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS) were used for the charac- terization of atherosclerotic plaques. Our NLO-based image analysis of animal arteries affected by atherosclerotic plaque accumulation revealed that images of the healthy regions of the artery can be readily distinguished by marked differences in morphology, due to a fluorescent signal generated from the presence of generally intact elastic layer. Regions affected by lesions were dominated by lipid-rich cells and collagen fibers; the elastic layer was disrupted and the presence of fluorescent particles were also detected. Next, the potential of using information extracted from NLO images lead us to the development of a new optical index for plaque burden (OIPB). Through the OIPB, it was possible to investigate and to classify the plaque severity regarding the already established and currently used definition during clinical analyses. Extrapolating to and anticipating future applications, several methods for extracting specific information from images acquired by each NLOM modality were developed and tested. Texture analysis, particle-specific features, fractal analysis and directionality of components within the images were successfully adapted and tailored to better extract relevant information from the NLO images. Even though the methods presented in this thesis were mostly tested in images from arterial plaques, there is strong evidence that all tools presented here are capable of tracking changes that occur in many medical conditions and applications.

Visame pasaulyje skrandžio vėžys yra didelė problema, kasmet diagnozuojama apie 875000 naujų atvejų ir 645000 miršta nuo šios ligos. Lietuvos vėžio registro duomenimis, 2005 m. Lietuvoje sergamumas skrandžio vėžiu buvo 35,0 vyrų ir 22,1 moterų tarpe 100000 gyventojų. Šis susirgimas vis dar dažniausiai diagnozuojamas vėlyvose stadijose, kuomet radikali operacija jau nėra galima. Skrandžio vėžio vystymasis yra kompleksinis ir šiuo metu dar pilnai neišaiškintas procesas. Nauji tyrimai patvirtino, kad epigenetinė pažaida – TPEF/HPP1 geno metilinimas yra dažnas reiškinys tulžies ir šlapimo pūsles, kolorektalinio vėžio atvejais. Vis dėlto, nebuvo pakankamai duomenų apie TPEF/HPP1 geno metilinimo reikšmę skrandžio kancerogenezėje. Atliktame tyrime nustatyta, kad TPEF/HPP1 geno metilinimas gali būti ankstyvas šio pažeidimo požymis. Taip pat galima daryti prielaidą, kad TPEF/HPP1 genas yra skrandžio naviką slopinantis genas. Kitas svarbus skrandžio vėžio rizikos faktorius yra H. pylori sąlygotas atrofinis gastritas. Tyrimo metu nustatytas didelis vyresnių nei 55 m. dispepsija sergančių pacientų infekuotumas H. pylori. Statistiškai patikimo skirtumo tarp atrofijos ir žarninės metaplazijos dažnumo dispepsija sergantiems pacientams Taivanyje, Lietuvoje, Latvijoje negauta. Buvo nustatyta stipri koreliacija tarp skrandžio atrofijos ir žarninės metaplazijos. Klinikinėje praktikoje atrofinis gastritas patvirtinamas histologiškai (pagal 1994 m. Hiūstone modifikuotą Sidnėjaus klasifikaciją)... [toliau žr. visą tekstą] / INTRODUCTION Gastric cancer is rampant in many countries around the world and it accounts for approximately 875000 new cases and 645000 deaths annually [Jemal A et al, 2004]. While overall incidence of gastric cancer is falling, in many countries of the world it remains one the most frequent causes of cancer related deaths. According to GLOBOCAN age-standardized cancer incidence data, in Germany 15.1 males and 8.8 females per 100.000 persons developed gastric cancer in 2002, in Lithuania – 25.3 males and 13.0 females, in Latvia – 24.6 males and 11.1 females, respectively. According to the data of Lithuanian Cancer Registry, gastric cancer incidence was 35.0 in male, 22.1 in female per 100.000 persons in 2005, 30.4 in male, 17.8 in female in 2004, respectively [Kurtinaitis J, 2004]. At present, primary or secondary prevention is likely to be the most effective means of reducing the incidence and mortality from this disease. However, to be successful, this strategy depends upon knowledge of the etiological factors and pathogenetic mechanisms involved in gastric carcinogenesis. Helicobacter pylori (H. pylori) has been categorized as a group I carcinogen by the International Agency for Research on Cancer and World Health Organization (WHO) in 1994. Development of gastric cancer is a complex and poorly understood process. It is clear that besides chronic gastritis caused by H. pylori, dietary factors, high salt and nitrate intake, smoking and, possibly, alcohol consumption are... [to full text]

Le travail présenté dans ce mémoire de thèse avait pour but de synthétiser de nouveaux composés pour des applications en imagerie médicale. La première partie porte sur la synthèse de ligands hétéroleptiques pour la chélation de deux métaux différents en vue d’une utilisation dans deux types d’imagerie. Pour cela, nous avons d’abord synthétisé des porphyrines. Ces molécules sont connues pour la chélation de nombreux métaux de transition et notamment le cuivre, dont l’isotope cuivre-64 est un radioémetteur β+ utilisable en Tomographie par Émission de Positron (TEP). Ces porphyrines ont été couplées à un dérivé du DOTA, molécule connue pour son application, après métallation avec du gadolinium, en Imagerie par Résonance Magnétique (IRM). Ces composés possèdent des valeurs de relaxivité encourageantes pour une application en IRM. Enfin, des biomolécules ont été modifiées afin de vectoriser nos composés. La seconde partie porte sur la synthèse de composés pour l’imagerie médicale par optique non-linéaire. Nous avons dans un premier temps synthétisé des porphyrines amphiphiles et zwiterrioniques pour la Génération de Second Harmonique (GSH). Leur efficacité a été mesurée et celle-ci est suffisante pour pouvoir envisager l’obtention d’images. Dans un second temps, nous avons travaillé sur la synthèse d’un composé pour une application en imagerie biphotonique et en IRM. Pour cela, nous avons relié un dérivé du DOTA avec un fluorophore de type DPP. Le composé final a été métallé avec du gadolinium et sa relaxivité est supérieure à celle du DOTA(Gd). / The goal of my PhD studies was to synthesize new compounds for possible medical imaging applications. The first part of my thesis focused on the synthesis of heteroleptic ligands to achieve the chelation of two different metals aimed at addressing two types of medical imaging. We first synthesized porphyrins, which are well-known for the chelation of numerous transition metals. We focused on copper, whose copper-64 isotope is a β+ emitter usable in Positron Emission Tomography (PET). These porphyrins have been coupled with a DOTA derivative. This molecule, metallated with gadolinium, is well-known in Magnetic Resonance Imaging (MRI). Our compounds display encouraging relaxivities for MRI applications. At last, these molecular probes have been conjugated to a few biomolecules in order to vectorize our compounds. The second part of this work is related to the synthesis of fluorophores for nonlinear optical imaging. We first synthesized amphiphilic zwitterionic porphyrins for Second Harmonic Generation (SHG). The efficiency of these compounds has been measured and was satisying enough to consider the possibility to perform imaging studies. We also worked on the synthesis of compounds for Two Photon Emission Fluorescence (TPEF) imaging and MRI. We have linked a DOTA derivative with a diketopyrrolopyrrole (DPP). This conjugate has been metallated with gadolinium and its relaxivity has been measured. Interestingly, this value is superior to that of DOTA(Gd).

Optics are a powerful probe of chemical structure that can often be linked to theoretical predictions, providing robustness as a measurement tool. Not only do optical interactions like second harmonic generation (SHG), single and two-photon excited fluorescence (TPEF), and infrared absorption provide chemical specificity at the molecular and macromolecular scale, but the ability to image enables mapping heterogeneous behavior across complex systems such as biological tissue. This thesis will discuss nonlinear and linear optics, leveraging theoretical predictions to provide frameworks for interpreting analytical measurement. In turn, the causal mechanistic understanding provided by these frameworks will enable structurally specific quantitative tools with a special emphasis on application in biological imaging. The thesis will begin with an introduction to 2nd order nonlinear optics and the polarization analysis thereof, covering both the Jones framework for polarization analysis and the design of experiment. Novel experimental architectures aimed at reducing 1/f noise in polarization analysis will be discussed, leveraging both rapid modulation in time through electro-optic modulators (Chapter 2), as well as fixed-optic spatial modulation approaches (Chapter 3). In addition, challenges in polarization-dependent imaging within turbid systems will be addressed with the discussion of a theoretical framework to model SHG occurring from unpolarized light (Chapter 4). The application of this framework to thick tissue imaging for analysis of collagen local structure can provide a method for characterizing changes in tissue morphology associated with some common cancers (Chapter 5). In addition to discussion of nonlinear optical phenomena, a novel mechanism for electric dipole allowed fluorescence-detected circular dichroism will be introduced (Chapter 6). Tackling challenges associated with label-free chemically specific imaging, the construction of a novel infrared hyperspectral microscope for chemical classification in complex mixtures will be presented (Chapter 7). The thesis will conclude with a discussion of the inherent disadvantages in taking the traditional paradigm of modeling and measuring chemistry separately and provide the multi-agent consensus equilibrium (MACE) framework as an alternative to the classic meet-in-the-middle approach (Chapter 8). Spanning topics from pure theoretical descriptions of light-matter interaction to full experimental work, this thesis aims to unify these two fronts. <br>