Confocal Microscopy and Nuclear Segmentation Algorithm for Quantitative Imaging of Epithelial Tissue

Carcinomas, cancers that originate in the epithelium, account for more than 80% of all cancers. When detected early, the 5-year survival rate is greatly increased. Biopsy and histopathology is the current gold standard for diagnosis of epithelial carcinomas which is an invasive, time-intensive, and stressful procedure. In vivo confocal microscopy has the potential to non-invasively image epithelial tissue in near-real time. This dissertation describes the development of a confocal microscope for imaging epithelial tissues and an image processing algorithm for segmentation of epithelial nuclei. A rapid beam and stage scanning combination was used to acquire fluorescence confocal images of cellular and tissue features along the length of excised mouse colon. A single 1 × 60 mm2 field of view is acquired in 10 seconds. Disruption of crypt structure such as size, shape, and distribution is visualized in images of inflamed colon tissue, while the normal mouse colon exhibited uniform crypt structure and distribution. An automated pulse coupled neural network segmentation algorithm was developed for epithelial nuclei segmentation. An increase in nuclear size and the nuclear-to-cytoplasmic ratio is a potential precursor to pre-cancer development. The spiking cortical model algorithm was evaluated using a developed confocal image model of epithelial tissues with varying contrast. It was further validated on reflectance confocal images of porcine and human oral tissue from two separate confocal imaging systems. Biopsies of human oral mucosa are used to determine the tissue and system effects on measurements of nuclear-to-cytoplasmic ratio

Morphological Features of Dysplastic Progression in Epithelium: Quantification of Cytological, Microendoscopic, and Second Harmonic Generation Images

Advances in imaging technology have led to a variety of available clinical and investigational systems. In this collection of studies, we tested the relevance of morphological image feature quantification on several imaging systems and epithelial tissues. Quantification carries the benefit of creating numerical baselines and thresholds of healthy and abnormal tissues, to potentially aid clinicians in determining a diagnosis, as well as providing researchers with standardized, unbiased results for future dissemination and comparison. Morphological image features in proflavine stained oral cells were compared qualitatively to traditional Giemsa stained cells, and then we quantified the nuclear to cytoplasm ratio. We determined that quantification of proflavine stained cells matched our hypothesis, as the nuclei in oral carcinoma cells were significantly larger than healthy oral cells. Proflavine has been used in conjunction with translational fluorescence microendoscopy of the gastrointestinal tract, and we demonstrated the ability of our custom algorithm to accurately (up to 85% sensitivity) extract colorectal crypt area and circularity data, which could minimize the burden of training on clinicians. In addition, we proposed fluorescein as an alternative fluorescent dye, providing comparable crypt area and circularity information. In order to investigate the morphological changes of crypts via the supporting collagen structures, we adapted our quantification algorithm to analyze crypt area, circularity, and an additional shape parameter in second harmonic generation images of label-free freshly resected murine epithelium. Murine models of colorectal cancer (CRC) were imaged at early and late stages of tumor progression, and we noted significant differences between the Control groups and the late cancer stages, with some differences between early and late stages of CRC progression

Strain-induced alignment in collagen gels

Collagen is the most abundant extracellular-network-forming protein in animal biology and is important in both natural and artificial tissues, where it serves as a material of great mechanical versatility. This versatility arises from its almost unique ability to remodel under applied loads into anisotropic and inhomogeneous structures. To explore the origins of this property, we develop a set of analysis tools and a novel experimental setup that probes the mechanical response of fibrous networks in a geometry that mimics a typical deformation profile imposed by cells in vivo. We observe strong fiber alignment and densification as a function of applied strain for both uncrosslinked and crosslinked collagenous networks. This alignment is found to be irreversibly imprinted in uncrosslinked collagen networks, suggesting a simple mechanism for tissue organization at the microscale. However, crosslinked networks display similar fiber alignment and the same geometrical properties as uncrosslinked gels, but with full reversibility. Plasticity is therefore not required to align fibers. On the contrary, our data show that this effect is part of the fundamental non-linear properties of fibrous biological networks.Comment: 12 pages, 7 figures. 1 supporting material PDF with 2 figure

Objective localisation of oral mucosal lesions using optical coherence tomography.

PhDIdentification of the most representative location for biopsy is critical in establishing the definitive diagnosis of oral mucosal lesions. Currently, this process involves visual evaluation of the colour characteristics of tissue aided by topical application of contrast enhancing agents. Although, this approach is widely practiced, it remains limited by its lack of objectivity in identifying and delineating suspicious areas for biopsy. To overcome this drawback there is a need to introduce a technique that would provide macroscopic guidance based on microscopic imaging and analysis. Optical Coherence Tomography is an emerging high resolution biomedical imaging modality that can potentially be used as an in vivo tool for selection of the most appropriate site for biopsy. This thesis investigates the use of OCT for qualitative and quantitative mapping of oral mucosal lesions. Feasibility studies were performed on patient biopsy samples prior to histopathological processing using a commercial OCT microscope. Qualitative imaging results examining a variety of normal, benign, inflammatory and premalignant lesions of the oral mucosa will be presented. Furthermore, the identification and utilisation of a common quantifiable parameter in OCT and histology of images of normal and dysplastic oral epithelium will be explored thus ensuring objective and reproducible mapping of the progression of oral carcinogenesis. Finally, the selection of the most representative biopsy site of oral epithelial dysplasia would be investigated using a novel approach, scattering attenuation microscopy. It is hoped this approach may help convey more clinical meaning than the conventional visualisation of OCT images

FIBER-OPTIC BUNDLE FLUORESCENCE MICROSCOPY FOR FUNCTIONAL BRAIN ACTIVITY MAPPING

Understanding the relationship between cellular activities in the animal brain and the emerging patterns of animal behavior is a critical step toward completing the Brain Activity Map. This dissertation describes the development of fiber-bundle microscopy capable of high-resolution cellular imaging, for mapping of functional brain activity in freely moving mice. As a part of this work, several fiber-bundle microscope systems and image processing algorithms were proposed and developed. These optical imaging methods and system performance were tested and evaluated by performing in vivo animal brain imaging. Several fiber-bundle imaging devices, including a dual-mode confocal reflectance and fluorescence micro-endoscope, a single ball-lens imaging probe, and a spatially multiplexed fiber-bundle imager, were designed and developed for high-resolution imaging of brain cells and visualization of brain activity. A dual-mode micro-endoscope, simultaneously achieving laser scanning confocal reflectance and fluorescence imaging, was developed to quantitatively assess gene transfection efficacy using human cervical cancer cells. A single ball-lens integrated imaging probe was designed for endoscopic brain imaging. Lastly, a spatially multiplexed fiber-bundle imager that allows concurrent monitoring of astrocytic activities in multiple brain regions and enables optical manipulation with cell-specific targeting was proposed and experimentally demonstrated. Novel image-processing algorithms were used along with the developed imaging systems. Structured illumination employing a digital micro-mirror device (DMD) was integrated into the system to achieve depth-resolved imaging with a wide-field illumination fiber-bundle microscope. Data from super-resolution fiber-bundle microscopy based on the linear structured illumination were numerically processed to extend the lateral resolution beyond the diffraction limit. To evaluate the performance of the developed fiber-bundle microscope systems and image reconstruction algorithms, the systems and methods were each tested and validated on in vivo animal models, namely transgenic mice expressing a genetically encoded Calcium indicator (GCaMP3) within astrocytes. We showed that locomotion triggers simultaneous activation of astrocyte networks in multiple brain regions in mice. We have also demonstrated real-time cellular-resolution dual-color functional brain imaging in mice. Finally, we established a platform that allows real-time and non-invasive imaging of the intact central nervous system of freely behaving mice. Using this platform, we observed, for the first time, physiologically relevant activation of astrocytes during behaviorally relevant tasks and in the natural setting. In addition, we present a proof-of-concept study by using a fiber-bundle ring light-guided portable multispectral imaging (MSI) platform capable of tissue characterization and preoperative surgical planning for intestinal anastomosis