An Investigation of the Diagnostic Potential of Autofluorescence Lifetime Spectroscopy and Imaging for Label-Free Contrast of Disease

The work presented in this thesis aimed to study the application of fluorescence lifetime spectroscopy (FLS) and fluorescence lifetime imaging microscopy (FLIM) to investigate their potential for diagnostic contrast of diseased tissue with a particular emphasis on autofluorescence (AF) measurements of gastrointestinal (GI) disease. Initially, an ex vivo study utilising confocal FLIM was undertaken with 420 nm excitation to characterise the fluorescence lifetime (FL) images obtained from 71 GI samples from 35 patients. A significant decrease in FL was observed between normal colon and polyps (p = 0.024), and normal colon and inflammatory bowel disease (IBD) (p = 0.015). Confocal FLIM was also performed on 23 bladder samples. A longer, although not significant, FL for cancer was observed, in paired specimens (n = 5) instilled with a photosensitizer. The first in vivo study was a clinical investigation of skin cancer using a fibre-optic FL spectrofluorometer and involved the interrogation of 27 lesions from 25 patients. A significant decrease in the FL of basal cell carcinomas compared to healthy tissue was observed (p = 0.002) with 445 nm excitation. A novel clinically viable FLS fibre-optic probe was then applied ex vivo to measure 60 samples collected from 23 patients. In a paired analysis of neoplastic polyps and normal colon obtained from the same region of the colon in the same patient (n = 12), a significant decrease in FL was observed (p = 0.021) with 435 nm excitation. In contrast, with 375 nm excitation, the mean FL of IBD specimens (n = 4) was found to be longer than that of normal tissue, although not statistically significant. Finally, the FLS system was applied in vivo in 17 patients, with initial data indicating that 435 nm excitation results in AF lifetimes that are broadly consistent with ex vivo studies, although no diagnostically significant differences were observed in the signals obtained in vivo.Open Acces

Swept source optical coherence microscopy for pathological assessment of cancerous tissues

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Optical coherence microscopy (OCM) combines optical coherence tomography (OCT) with confocal microscopy and enables depth resolved visualization of biological specimens with cellular resolution. OCM offers a suitable alternative to confocal imaging by providing enhanced contrast due to the additional coherence gate to the inherent confocal gate, increasing the field of view and imaging depth, and eliminating the need of external contrast agents. In the past, development of OCT systems have been focused on time domain and spectral/Fourier domain methods which offer high axial resolution and imaging speeds. However, recent advances in the OCT technology have pushed the development into the direction of swept source OCT technologies, and development of the OCM technology is likely to follow this path. This thesis describes construction, characterization and preliminary imaging results of a swept source OCM (SS-OCM) system utilizing a novel light source, Vertical Cavity Surface-Emission Laser (VCSEL). This swept source laser can reach sweep rates exceeding 1 MHz and provide wide tuning ranges, which will enable both imaging speeds approaching to time domain OCM (TD-OCM) systems, and axial resolution approaching to spectral/Fourier domain OCM (SD-OCM) systems. Several other advantages of SS-OCM compared to TD-OCM and SD-OCM that make this technology a promising alternative to the latter imaging methods are presented. Furthermore, practical concepts in the system development and signal processing, such as compensation for the scan curvatures, methods for calibration of the spectrums, selection of suitable color maps for display, and other related topics are also discussed in the text. In addition to technical description of the OCM system development, an in depth analysis of several clinical applications that will be likely to benefit from this imaging modality is also presented. Real time intraoperative feedback is required in order to reduce the morbidity and the rate of additional operations for the surgical management of several forms of cancer, where a benchtop OCM system residing in the pathology laboratory can be immensely beneficial. Furthermore, with the novel scanning mechanisms that have been developed in the recent years it is possible to translate this imaging modality to an in vivo setting where an OCM probe can be inserted through the working channel of an endoscope and generate cellular resolution images in real time without the need of external contrast agents. Endoscopic management and clinical challenges for a spectrum of lower gastrointestinal (GI) diseases is discussed where an in vivo OCM imaging probe can play an important role in the diagnosis and evaluation of the extend of the particular disease. A review of alternative imaging modalities, such as chromoendoscopy, narrow band imaging (NBI) and confocal laser endomicroscopy (CLE) is also included which outlines the relative strengths and limitations of these imaging modalities for the clinical management of lower GI diseases.by Osman Oguz Ahsen.S.M

Endoscopic optical coherence tomography for clinical studies in the gastrointestinal tract

Thesis (Ph. D.)--Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Optical coherence tomography (OCT) performs micrometer-scale, cross-sectional and three dimensional imaging by measuring the echo time delay of backscattered light. OCT imaging is performed using low-coherence interferometry. With the development of Fourier domain detection techniques and fiber-optic based OCT endoscopes, high speed internal body imaging was enabled, which makes OCT suitable for clinical research in the human gastrointestinal (GI) tract. Endoscopic OCT imaging is challenging because fast and stable optical scanning must be implemented inside a small imaging probe to acquire useable volumetric information from internal human bodies. Although several studies have shown the use of endoscopic OCT in human gastrointestinal tracts as a real-time surveillance tool, the capability of OCT has not yet been fully explored in endoscopic applications and OCT is not well accepted as a standard imaging modality for GI clinics due to hardware limitations and lack of comprehensive clinical evidences. This thesis presents a number of clinical studies using endoscopic OCT that provide solutions to clinical problems in the GI tract supported by statistically significant results and the development of ultrahigh speed endoscopic OCT system that enables advanced OCT imaging applications. In collaboration with medical partners, the structural features in the diseased esophagus identified from OCT images are compared before and immediately after different ablative therapies, and features that predict the treatment response are investigated. Working in collaboration with industrial partners, an ultrahigh speed endoscopic OCT imaging system is constructed for clinical research in gastroenterology. Distally actuated imaging catheters are developed, enabling the visualization of the detailed three-dimensional (3D) structure in the gastrointestinal tract. Finally, clinical pilot studies are conducted and demonstrate the utility of the ultrahigh speed endoscopic OCT imaging for broader surveillance coverage, pathology detection, and dye-less contrast enhancement. The convergence of 3D spatial resolution, imaging speed, field of view, and minimally invasive access enabled by endoscopic OCT are unmatched by most other biomedical imaging techniques. Though still in its early stage of clinical validation, endoscopic OCT may have a profound impact on human healthcare and industrial inspection by enabling visualization and quantification of 3D microstructure in situ and in real time.by Tsung-Han Tsai.Ph.D

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

TOWARDS HIGH RESOLUTION ENDOSCOPIC OPTICAL COHERENCE TOMOGRAPHY FOR IMAGING INTERNAL ORGANS

Optical coherence tomography (OCT) is a light based interferometric imaging technique that can provide high resolution (5-20 µm at 1300 nm), depth resolved, images in real-time. With recent advances in portable low coherent light sources for OCT it is now possible to achieve ultrahigh axial resolutions (≤ 3 µm) by moving to shorter central wavelengths such as 800 nm while utilizing a broad spectral bandwidth. Our goal was to push ultrahigh resolution OCT technology to in vivo imaging of internal organs for endoscopic assessment of tissue microstructure. This dissertation is separated into technological developments and biomedical imaging studies. Technological developments in this dissertation included development of a high speed, ultrahigh resolution distal scanning catheter. This catheter was based upon a miniature DC micromotor capable of rotational velocities in excess of 100 rps, a diffractive compound lens design that minimized chromatic aberrations, and a mechanical assembly that limited field of view blockage to less than 7.5% and maintained an outer diameter of 1.78 mm (with plastic sheath). In conjunction with the algorithm described in chapter 4 to correct for non-uniform rotational distortion, the overall imaging system was capable of high quality endoscopic imaging of internal organs in vivo. Equipped with the ultrahigh resolution endoscopic OCT system, imaging was performed in small airways and colorectal cancer. Imaging results demonstrated the ability to directly visualization of microstructural details such as airway smooth muscle in the small airways representing a major step forward in pulmonary imaging. With the ability to visualize airway smooth muscle, morphological changes in COPD and related diseases can be further investigated. Additionally, longitudinal changes in an ETBF induced colon cancer model in APCMin mice were studied as well. Quantitative assessment of tissue microarchitecture was performed by measuring the attenuation coefficient to find a bimodal distribution separating normal healthy tissue from polyps. Finally, results from two additional projects were also demonstrated. Chapter 7 shows some results from vascular imaging in a tumor angiogenesis model and middle cerebral artery occlusion model. Chapter 8 describes an endoscopic multimodal OCT and fluorescence imaging platform with results in ex vivo rabbit esophagus

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

A deep-learning approach to aid in diagnosing Barrett’s oesophagus related dysplasia

Barrett's oesophagus is the only known precursor to oesophagus carcinoma. Histologically, it is defined as a condition of columnar cells replacing the standard squamous lining. Those altered cells are prone to cytological and architectural abnormalities, known as dysplasia. The dysplastic degree varies from low to high grade and can evolve into invasive carcinoma or adenocarcinoma. Thus, detecting high-grade and intramucosal carcinoma during the surveillance of Barrett's oesophagus patients is vital so they can be treated by surgical resection. Unfortunately, the achieved interobserver agreement for grading dysplasia among pathologists is only fair to moderate. Nowadays, grading Barrett's dysplasia is limited to visual examination by pathologists for glass or virtual slides. This work aims to diagnose different grades of dysplasia in Barrett’s oesophagus, particularly high-grade dysplasia, from virtual histopathological slides of oesophagus tissue. In the first approach, virtual slides were analysed at a low magnification to detect regions of interest and predict the grade of dysplasia based on the analysis of the virtual slides at 10X magnification. Transfer learning was employed to partially fine-tune two deep-learning networks using healthy and Barrett’s oesophagus tissue. Then, the two networks were connected. The proposed model achieved 0.57 sensitivity, 0.79 specificity and moderate agreement with a pathologist. On the contrary, the second approach processed the slides at a higher magnification (40X magnification). It adapted novelty detection and local outlier factor alongside transfer learning to solve the multiple instances learning problem. It increased the performance of the diagnosis to 0.84 sensitivity and 0.92 specificity, and the interobserver agreement reached a substantial level. Finally, the last approach mimics the pathologists’ procedure to diagnose dysplasia, relying on both magnifications. Thus, their behaviours during the assessment were analysed. As a result, it was found that employing a multi-scale approach to detect dysplastic tissue using a low magnification level (10X magnification) and grade dysplasia at a higher level (40X magnification). The proposed computer-aided diagnosis system was built using networks from the first two approaches. It scored 0.90 sensitivity, 0.94 specificity and a substantial agreement with the pathologist and a moderate agreement with the other expert