Multimodal Multispectral Optical Endoscopic Imaging for Biomedical Applications

Optical imaging is an emerging field of clinical diagnostics that can address the growing medical need for early cancer detection and diagnosis. Various human cancers are amenable to better prognosis and patient survival if found and treated during early disease onset. Besides providing wide-field, macroscopic diagnostic information similar to existing clinical imaging techniques, optical imaging modalities have the added advantage of microscopic, high resolution cellular-level imaging from in vivo tissues in real time. This comprehensive imaging approach to cancer detection and the possibility of performing an ‘optical biopsy’ without tissue removal has led to growing interest in the field with numerous techniques under investigation. Three optical techniques are discussed in this thesis, namely multispectral fluorescence imaging (MFI), hyperspectral reflectance imaging (HRI) and fluorescence confocal endomicroscopy (FCE). MFI and HRI are novel endoscopic imaging-based extensions of single point detection techniques, such as laser induced fluorescence spectroscopy and diffuse reflectance spectroscopy. This results in the acquisition of spectral data in an intuitive imaging format that allows for quantitative evaluation of tissue disease states. We demonstrate MFI and HRI on fluorophores, tissue phantoms and ex vivo tissues and present the results as an RGB colour image for more intuitive assessment. This follows dimensionality reduction of the acquired spectral data with a fixed-reference isomap diagnostic algorithm to extract only the most meaningful data parameters. FCE is a probe-based point imaging technique offering confocal detection in vivo with almost histology-grade images. We perform FCE imaging on chemotherapy-treated in vitro human ovarian cancer cells, ex vivo human cancer tissues and photosensitiser-treated in vivo murine tumours to show the enhanced detection capabilities of the technique. Finally, the three modalities are applied in combination to demonstrate an optical viewfinder approach as a possible minimally-invasive imaging method for early cancer detection and diagnosis

Fast widefield techniques for fluorescence and phase endomicroscopy

Thesis (Ph.D.)--Boston UniversityEndomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast widefield imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated

Assessing Molecular Biomarkers in Living Mice Using Fluorescence Microendoscopy and Spectroscopy.

Assessment of molecular biomarkers expressed in cells and tissues can inform scientists and clinicians of physiological and disease processes. Optical techniques can quantitatively and noninvasively assess molecular biomarkers in living tissues. This dramatically improves our ability to study detailed behavior of disease, perform earlier detection of disease, and assess functional cellular information. However, small animals, which play an important role in the study of molecular biomarkers, pose a challenge for intravital optical assessment. In this dissertation, we engineer and demonstrate methodologies for performing intravital optical assessments, in living mice, of fluorescent biomarkers that indicate molecular expressions of disease or viability. First, we engineered a flexible fiber-optic microendoscope for longitudinal optical imaging studies in a mouse model of disseminated ovarian cancer. This microendoscope has an outer diameter of 680 µm and achieves a lateral resolution of 4 µm. The instrument repetitively monitored the growth of fluorescence-expressing ovarian cancer cells in mice for over 4 weeks, visualizing single cells, cell clusters, and tumor masses. By establishing longitudinal (non-terminal) studies, this technology allows each animal to be used as its own control, significantly reducing the number of animals needed for experimentation. We then employed fluorescence microendoscopy to validate the specific binding activity of a fluorescence-labeled peptide to colorectal dysplasia in a genetically-engineered mouse model. The microendoscope was passed through the instrument channel of a small animal endoscope for simultaneous wide-field and microscopic imaging. More than two-fold greater fluorescence intensity was measured from dysplastic tissue compared to adjacent normal mucosa. In the third part of this dissertation we developed a label-free methodology employing a handheld fluorescence lifetime spectroscopy probe to optically assess tissue engineered constructs that were implanted in living mice. Clinical translation of tissue engineered constructs requires noninvasive methods for assessing their integration with host tissue after grafting. Our instrumentation noninvasively sensed endogenous fluorophores in the tissue constructs that correlate to in vitro measures of cellular viability. Finally, we report the design and construction of a depth-resolved fluorescence lifetime spectroscopy system, which could be used for assessing the viability of tissue-engineered constructs with greater specificity than the demonstrated probe.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108845/1/sfelahi_1.pd

Clinical Reflectance Confocal Microscope for Imaging of Oral Cancer

Biopsy and histopathology remain the standard method for diagnosis of oral cancer in the clinic today. Early detection of oral cancer is fundamental to a higher survival rate, and a non-invasive method is preferred. This is possible through optical imaging techniques. This dissertation describes the design, development and testing of a clinical reflectance confocal microscope for imaging of oral cancer in combination with macroscopic fluorescence lifetime imaging (FLIM). A compact bench top reflectance confocal microscope was designed and constructed for use in combination with a bench top FLIM system. The system was evaluated by imaging porcine oral tissue ex vivo and normal and dysplastic hamster cheek pouch tissue in vivo. To facilitate in vivo imaging of the human oral cavity, an electrically tunable lens was integrated in the system for axial scanning and a miniature objective lens was designed and fabricated for access into the oral cavity. Performance of the system was characterized over the full range of axial scanning with the electrically tunable lens. The reflectance confocal microscopy system was tested in combination with macroscopic FLIM by imaging normal and pre-cancerous human oral tissue ex vivo and in vivo in the clinic

High-resolution imaging for cancer detection with a fiber bundle microendoscope

Dysplasia and cancer of epithelial tissues, including the oral cavity and esophagus, typically have much higher survival rates if diagnosed at an early stage. Unfortunately, the clinical appearance of lesions in these tissues can be highly variable. To achieve a definitive diagnosis of a suspected lesion at these sites, an excisional biopsy must be examined at high-resolution. These procedures can be costly and timeconsuming, and in the case of Barrett's esophagus, surveillance biopsy strategies may not be entirely effective. Optical imaging modalities have the potential to yield qualitative and quantitative high-resolution data at low cost, enabling clinicians to improve early detection rate. This dissertation presents a low-cost high-resolution microendoscopy system based on a fiber optic bundle image guide. In combination with a topical fluorescent dye, the fiber bundle can be placed into contact with the tissue to be observed. A high-resolution image is then projected onto a CCD camera and stored on a PC. A pilot study was performed on both resected esophageal tissue containing intestinal metaplasia (a condition known as Barrett's esophagus, which can transform to esophageal adenocarcinoma) and resected oral tissue following surgical removal of cancer. Qualitative image analysis demonstrated similar features were visible in both microendoscope images and standard histology images, and quantitative image processing and analysis yielded an objective classification algorithm. The classification algorithm was developed to discriminate between neoplastic and non-neoplastic imaging sites. The performance of this algorithm was monitored by comparing the predicted results to the pathology diagnosis at each measurement site. In the oral cancer pilot study, the classifier achieved 85% sensitivity and 78% specificity with 141 independent measurement sites. In the Barrett's metaplasia pilot study, 87% sensitivity and 85% specificity were achieved with 128 independent measurement sites. The work presented in this dissertation outlines the design, testing, and initial validation of the high-resolution microendoscope system. This microendoscope system has demonstrated potential utility over a wide range of modalities, including small animal imaging, molecular-specific imaging, ex vivo and ultimately in vivo imaging

Demonstrating the Use of Optical Fibres in Biomedical Sensing:A Collaborative Approach for Engagement and Education

This paper demonstrates how research at the intersection of physics, engineering, biology and medicine can be presented in an interactive and educational way to a non-scientific audience. Interdisciplinary research with a focus on prevalent diseases provides a relatable context that can be used to engage with the public. Respiratory diseases are significant contributors to avoidable morbidity and mortality and have a growing social and economic impact. With the aim of improving lung disease understanding, new techniques in fibre-based optical endomicroscopy have been recently developed. Here, we present a novel engagement activity that resembles a bench-to-bedside pathway. The activity comprises an inexpensive educational tool ($70) adapted from a clinical optical endomicroscopy system and tutorials that cover state-of-the-art research. The activity was co-created by high school science teachers and researchers in a collaborative way that can be implemented into any engagement development process

Using Fluorescence – Polarization Endoscopy in Detection of Precancerous and Cancerous Lesions in Colon and Pancreatic Cancer

Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real time detection of these lesions. Utilizing a new thermoresponsive sol-gel formulation with targeted molecular probe allowed topical application and detection of precancerous and cancerous lesions during endoscopy. Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy served as a validation strategy prior to obtaining biopsies. In order to reduce repeat surgeries arising from incomplete tumor resection, we demonstrated the efficacy of the targeted molecular probe towards margins of sporadic colorectal cancer (SCC). Fluorescence-polarization microscopy using circular polarized (CP) light served as a rapid, supplementary tool for assessment and validation of excised tissue to ensure complete tumor resection for examining tumor margins prior to H&E-based pathological diagnosis. We extended our platform towards non-invasive directed detection of pancreatic cancer utilizing fluorescence molecular tomography (FMT) and NIR laparoscopy using identified targeted molecular probe. We were able to non-invasively distinguished between pancreatitis and pancreatic cancer and guide pancreatic tumor resection using NIR laparoscopy

Multi-spectral Dual Axes Confocal Endomicroscope with Vertical Cross-sectional Scanning for In-vivo Targeted Imaging of Colorectal Cancer

Pathologists review histology cut perpendicular to the tissue surface or in the vertical cross-section (XZ-plane) in order to visualize the normal or abnormal differentiation patterns. The epithelium of hollow organs, such as the colon, is the origin of many important forms of cancer. The vertical cross-section provides a comprehensive view of the epithelium which normally differentiates in the basilar to luminal direction. Real-time imaging in this orientation has not been fully explored in endomicroscopy because most instruments collect images in the horizontal cross-section (XY-plane). Imaging microstructures from the tissue surface to about half a millimeter deep can reveal early signs of disease. Furthermore, the use of molecular probes is an important, emerging direction in diagnostic imaging that improves specificity for disease detection and reveals biological function. Dysplasia is a pre-malignant condition in the colon that can progress into colorectal cancer. Peptides have demonstrated tremendous potential for in-vivo use to detect colonic dysplasia. Moreover, peptides can be labeled with NIR dyes for visualizing the full depth of the epithelium in small animals. This study aims to demonstrate large FOV multi-spectral targeted in-vivo vertical optical section with a dual axes confocal endomicroscope enabled by MEMS technology. The NIR multi-spectral fluorescence images demonstrate both histology-like morphology imaging and molecular imaging of specific peptide binding to dysplasia in the mouse colon. The specific aims of this study are: (1) to develop miniature vertical cross-sectional scan engine based on MEMS technology for imaging on XZ-plane; (2) to integrate micro-optics and develop multi-spectral dual axes confocal endomicroscope imaging system; (3) to perform in-vivo targeted vertical cross-sectional imaging with large FOV on colorectal cancer mouse model.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107154/1/zqiu_1.pd

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