Confocal microscopy demonstrated its value in the diagnosis of Acanthamoeba and fungal keratitis which considered sight-threatening corneal diseases. However, it can be difficult to find and train confocal microscopy graders to accurately detect Acanthamoeba cysts and fungal filaments in the images. Use of an automated system could overcome this problem and help to start the correct treatment more quickly. Also, response to treatment can be difficult to assess in infectious keratitis using clinical examination alone, but there is evidence that the morphology of filaments and cysts may change over time with the use of correct treatment. An automated system to analyse confocal microscopy images for such changes would also assist clinicians in determining whether the ulcer is improving, or whether a change of treatment is needed. This research proposes a fully automated novel system with GUI to detect cysts and hyphae (filaments) and measure useful quantitative parameters for them through many stages; Image enhancement, image segmentation, quantitative analysis for detected cysts and hyphae, and registration and tracking of ordered sequence of images. The performance of the proposed segmentation procedure is evaluated by comparing between the manual and the automated traced images of the dataset that was provided by the Manchester Royal Eye Hospital. The positive predictive values rate of cysts for Acanthamoeba images was 76%. For detected hyphae in Fusarium images, many standard measurements were computed. The accuracy of their values was quantified by calculating the percent error rate for each measurement and which ranged from 23% to 49%.

Un déséquilibre dans les mécanismes de défense de la cornée peut entraîner une invasion par des micro-organismes (MO) et générer une kératite infectieuse (KI), ou encore un syndrome sec. Les KI sont la première cause de cécité monoculaire. Le syndrome sec, également fréquent, est sujet à de nombreuses études cliniques pour l’évaluation de nouveaux traitements. L’objectif de cette thèse est de mettre au point des méthodes d’imagerie pour le diagnostic et le suivi des pathologies de la surface oculaire au travers de deux projets innovants : 1/ le projet FLUOCOR consistant à développer une solution complète de diagnostic rapide in vivo des MO les plus souvent responsables des KI afin de débuter immédiatement le traitement adapté et améliorer le pronostic visuel final. Cette solution comprendra des BioMarqueurs fluorescents (BMfs) spécifiques des agents infectieux, fluorescents dans le rouge ou le proche infrarouge non éblouissant et un nouvel instrument optique de détection. Des BMfs basés sur le couplage de BODIPY innovant avec des molécules ciblant les MO ont été obtenus. Des tests in vitro ont montré une bonne spécificité mais les tests ex vivo sur cornées humaines ont montré une fixation non spécifique sur les cellules épithéliales. Pour pallier ce manque de spécificité, le couplage de ces BODIPY sur des anticorps dirigés contre les MO est en cours. 2/ le projet FLUOSCOPE qui a permis de développer une nouvelle stratégie d’imagerie de la surface oculaire pour le suivi du syndrome sec, de la méthode d’instillation des colorants jusqu’au traitement d’images, en passant par la conception d’un instrument optique désormais industrialisé par les laboratoires Théa. / An imbalance in the defence system of cornea can result in several diseases, like infectious keratitis (IK) in case of invasion by microorganisms (MO) or dry eye in case of insufficient tear quality and/or quality. IK are the first cause of unilateral blindness worldwide. Sicca syndrome, also frequent, is the subject of numerous clinical trials assessing new treatments. The aim of the PhD Thesis work is to develop new imaging methods for the diagnosis and follow-up of diseases of the ocular surface. Two innovative project were conducted: 1/ the FLUOCOR project consists in developing a complete solution of rapid in vivo etiologic diagnosis of IK, for the most frequent and/or severe infections to allow rapid starting of the most adapted treatment in order to improve the final visual prognosis. This solution will comprise fluorescent biomarkers (excitable by sustainable non blinding red or near infrared lights) specific to the target MO, and a new optical imaging device for their detection. Biomarkers based on new BODIPY coupled with molecules targeting the MO were obtained with a good in vitro specificity. Nevertheless, on ex vivo human corneas they stained superficial epithelial cells. In order to overcome this difficulty, the coupling of BODIPY on specific antibodies targeting the MO is ongoing; 2/ the FLUOSCOPE project that allowed development of a new strategy of ocular surface imaging for the diagnosis and follow up of sicca syndrome. The complete chain was revisited from the instillation of the dye to the objective quantification of staining by image analysis, through the development of a prototype of imaging device, now industrialized by les Laboratoires Thea.