Esophageal cancer is the eighth most common cancer in the world. Cancers of the esophagus account for 3.8% of all cases of cancers, with approximately 482,300 new cases reported in 2008 worldwide. In the United States alone, it is estimated that approximately 18,000 new cases will be diagnosed in 2013, and 15,210 deaths are expected. Despite advances in surgery and chemoradiation therapy, these advances have not led to a significant increase in survival rates, primarily because diagnosis often at an advanced and incurable stage when treatment is more difficult and less successful. Accurate, objective methods for early detection of esophageal neoplasia are needed. Here, quantitative classification algorithms for high resolution miscroendoscopic images were developed to distinguish between esophageal neoplastic and non-neoplastic tissue. A clinical study in 177 patients with esophageal squamous cell carcinoma (ESCC) was performed to evaluate the diagnostic performance of the classification algorithm in collaboration with the Mount Sinai Medical Center in the United States, the First Hospital of Jilin University in China, and the Cancer Institute and Hospital, the Chinese Academy of Medical Science in China. The study reported a sensitivity and specificity of 93% and 92%, respectively, in the training set, 87% and 97%, respectively, in the test set, and 84% and 95%, respectively, in an independent validation set. Another clinical study in 31 patients with Barrett’s esophagus resulted in a sensitivity of 84% and a specificity of 85%. Finally, a compact, portable version of the high resolution microendoscopy (HRME) device using a consumer-grade camera was developed and a series of biomedical experimental studies were carried out to assess the capability of the device.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, has an incubation period ranging from a few months to several years following infection via inhalation into the lungs. Whole body fluorescence scanners are used to image and monitor the growth of fluorescent protein expressing strains of M. tuberculosis in the lungs of animal models. Accurate quantitative analysis of bacterial growth during the early stages of infection inside lungs remains elusive, due to tissue absorption and scattering of photons emitted by the low numbers of bacteria deep in tissue. Fiber optic micro-endoscopy is uniquely suited to provide a novel solution to this problem by delivering light excitation directly to and collecting fluorescence from the infection site located in the lungs of an animal model, thereby enabling detection of fluorescent bacteria during the early stages of infection. In this thesis, I present a contact probe fiber bundle fluorescence micro-endoscope with a range of LED based excitation wavelengths, 4 μm resolution, a 750 μm field of view, and a 1 mm outer diameter. This system has detected tdTomato and GFP expressing Bacillus Calmette-Guérin (BCG) bacteria in vitro. Additionally, images of bacterial regions of infection obtained in mice subcutaneously infected with tdTomato expressing bacteria at concentrations ranging from 106 to 101 Colony Forming Units (CFU) and intra-tracheally infected mice at 106 CFU demonstrate the micro-endoscope’s capability to detect and resolve regions of bacterial infection in vivo. By relaying the bacterial fluorescence image from the infection site to an external detector, we are able to increase the sensitivity to early stages of infection.

This work presents several major developments related to a fluorescence confocal microendoscope technology that can provide instantaneous cellular level images from selected depths of tissue inside the human body. The confocal microendoscope systems discussed employ fiber-optic based imaging catheters coupled to custom built slit-scan confocal microscopes. One major new development involves the design, development, and testing of a new flexible confocal microgastroscope (CMG) system for imaging the esophagus. This new system has the potential to aid in the early detection of esophageal cancer. It consists of a new optical scan unit mounted on an endoscopy cart and a new flexible catheter that can be inserted through the instrument channel of a commercial gastroscope. The CMG system has higher spatial resolution and larger field of view than the previous generation clinical confocal microendoscopes in our lab. In addition, the new CMG system can be operated over a greater wavelength range than its predecessor. Central to the CMG system is the design, construction, and testing of a new distal miniature objective that enables high-quality microendoscopy. The miniature objective, built with all glass spherical surfaces, achieves diffraction-limited performance over a 486 to 1000 nm spectral range. The wide achromatic range of this lens allows the CMG system to be used with a variety of contrast agents including agents in the NIR region. In addition, the new miniature objective can be mounted on existing confocal microendoscopes in our lab such as the ovarian clinical confocal microlaparoscope and our laboratory based experimental system. Finally, a new confocal microlaparoscope with an articulating catheter capable of imaging inside the distal portion of fallopian tubes is presented. This instrument is intended to allow the detection of early stage ovarian cancer originating inside the fallopian tube. The new microlaparoscope is compatible with 5 mm trocars and includes a thin 2.2 mm diameter articulating distal tip consisting of a bare fiber bundle and an automated dye delivery system. The distal tip of this new endoscope can be articulated through simple wrist movements and locked in place at a given angle if desired. The thin distal tip and the ability to control the angle of the tip provide the size and flexibility needed to image inside the curved and delicate structures of the fallopian tube. Preliminary imaging results from the new CMG system, the achromatized miniature objective, and the new articulating confocal microlaparoscope are presented to demonstrate the performance and the potential of each system towards the overall goal of in vivo imaging and disease diagnosis.

Demographic data indicate that 60% of 6.7 million annual global cancer mortalities and 54% of 10.8 million new patients are in developing nations, unable or unwilling to avail of invasive screening tests that are the current norm. For most cancers, survival rate is strongly dependent on early detection, highlighting the need for improved screening methods. Studies have shown that cancers can be identified based on distinct sub-cellular morphological features and expression levels of specific molecular markers. Since 85% of cancers are known to originate in the epithelium, portable in vivo imaging techniques providing sub-cellular detail in tissue up to depths of 250 μm could help improve access to biopsy-free examination in low-infrastructure environments. The resultant early detection could dramatically improve patient prognosis, while reducing screening costs, treatment delay, and occurrences of unnecessary and potentially harmful medication. This dissertation investigates handheld instrumentation for laser-scanning confocal microscopy (LSCM) and its applicability to breast cancer detection and subsequent image-guided management. LSCM allows high-resolution mapping of spatial variations in refractive index or tumor marker expression within a single cell layer situated few hundred micrometers beneath the tissue surface. The main challenge facing miniaturization lies in the mechanism of beam deflection across the sample. The first part of the dissertation presents a fast, large-angle, high-reflectivity two-axis vertical comb driven silicon micromirror fabricated by a novel method compatible with complementary metal-oxide-semiconductor processing employed in the semiconductor industry. The process enables integration of rotation sensors on the chip to adaptively correct for aberrations in beam scanning while significantly reducing fabrication costs and barriers to market acceptance. The second part of the dissertation explores the integration of this micromirror with other optical and electronic components into a handheld laser-scanning confocal microscope. Applicability of the probe to epithelial breast cancer screening via reflectance and fluorescence imaging is investigated. Finally, enhanced imaging modalities based on the micromirror are presented. 3D cellular-level in vivo imaging via rapid swept-source optical coherence tomography is demonstrated. A method for “objective-less” microendoscopy, potentially resulting in substantially reduced probe dimensions, employing reflective binary-phase Fresnel zone plates monolithically integrated on the surface of the micromirror is presented. / text

Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (~1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography data. However, the multi-mode characteristic of the fibers in the fiber-bundle affects the depth sensitivity of the imaging system. A description of light interference in a multi-mode fiber is presented along with numerical simulations and experimental studies to illustrate the theoretical analysis.

Recent advances in label-free histology promise a new era for real-time diagnosis in neurosurgery. Deep learning using autofluorescence is promising for tumor classification without histochemical staining process. The high image resolution and minimally invasive diagnostics with negligible tissue damage is of great importance. The state of the art is raster scanning endoscopes, but the distal lens optics limits the size. Lensless fiber bundle endoscopy offers both small diameters of a few 100 microns and the suitability as single-use probes, which is beneficial in sterilization. The problem is the inherent honeycomb artifacts of coherent fiber bundles (CFB). For the first time, we demonstrate an end-to-end lensless fiber imaging with exploiting the near-field. The framework includes resolution enhancement and classification networks that use single-shot CFB images to provide both high-resolution imaging and tumor diagnosis. The well-trained resolution enhancement network not only recovers high-resolution features beyond the physical limitations of CFB, but also helps improving tumor recognition rate. Especially for glioblastoma, the resolution enhancement network helps increasing the classification accuracy from 90.8 to 95.6%. The novel technique enables histological real-time imaging with lensless fiber endoscopy and is promising for a quick and minimally invasive intraoperative treatment and cancer diagnosis in neurosurgery.

Multimódová vlákna jsou zobrazovacím prostředkem s významným potenciálem v in-vivo mikroendoskopii. V poslední době tato metoda zaznamenala velký rozvoj, a to díky zdokonalování výpočetní a jiné techniky, například prostorové modulace světla. Cílem této práce bylo nalézt specifické limity zobrazování multimódovými vlákny a představit jejich počítačovou simulaci. Byl zkoumán vliv způsobu osvětlení optického systému obsahujícího multimódové vlákno na jeho schopnost fokusace a zobrazování. Analýzou dat získaných ze simulací a experimentu bylo zjištěno, že různá míra omezení Gaussovského svazku a plnění apertury multimódového vlákna má za následek významnou změnu zobrazovacích schopností systému. Při pozorování kvality fokusace bylo zjištěno, že nejlépe se projevují svazky málo omezené aperturou vlákna. Tento fakt byl potvrzen i experimentálním měřením. Zobrazování za použití svazků s podobnými hodnotami omezení (50%) projevovalo i nejlepší schopnost přenosu kontrastu. Avšak při analýze rozlišení dvou bodových objektů se jako nejvhodnější projevily svazky významně přeplňující numerickou aperturu vlákna, 100% a více. Přítomnost tohoto rozdílu poukazuje na skutečnost, že multimódové vlákno není zcela náhodné médium, ale propagace světla skrz multimódové vlákno projevuje znaky závislosti na vnějších zobrazovacích podmínkách, jako je například změna omezení osvětlovacího svazku. V této práci bylo představeno několik způsobů vyhodnocení kvality zobrazování pomocí multimódového vlákna. Každé z těchto kritérií podalo dílčí charakteristiku chování optického systému obsahujícího multimódové optické vlákno. Jednotlivé výsledky se neshodují na jednom konkrétním řešení a nutí osobu využívající zobrazovací systém obsahující multimódové vlákno ke zvážení několika aspektů, a to v jakém prostředí bude daný optický systém využívat a který parametr kvality zobrazení bude považovat za nejdůležitější.

La microscopie multiphotonique est une modalité d’imagerie de pointe offrant des opportunités d’avancées remarquables en biologie mais aussi dans le domaine médical. Afin d’en exploiter pleinement le formidable potentiel au cœur même de la pratique clinique, le développement de nombreuses sondes miniaturisées à fibre optique pour l’endomicroscopie multiphotonique (EMMP) a eu lieu depuis de nombreuses années et dans de nombreux laboratoires français et étrangers. Il s’est pour l'instant confronté à des limitations majeures comme l’impossibilité de recueillir les signaux d’auto-fluorescence des tissus qui sont intrinsèquement faibles comme ceux venant des co-enzymes métaboliques NADH et FAD. Cette limitation compromet l'utilité de l’EMMP en la restreignant à une imagerie morphologique requérant un marquage exogène des tissus. Ce manuscrit présente une architecture d’EMMP permettant de dépasser cette limitation, capable de proposer une imagerie fonctionnelle du métabolisme cellulaire en temps réel, in vivo, in situ, sans marquage. Le prototype d’EMMP proposé est une amélioration du précédent, où les Grisms en réflexions sont remplacés par des Grisms en transmission, permettant d’élargir la bande spectrale d’utilisation et la transmission du système. Ce prototype voit aussi l’adjonction d’un second laser excitateur afin d’accéder aux fluorescences du NADH et du FAD. Les résultats démontrent capable que nous sommes à même d’imager les fluorescences cellulaires intrinsèques au travers de 5 mètres de fibre optique avec une résolution subcellulaire. Parmi celles-ci nous sommes capables d’exciter et de collecter spécifiquement les fluorescences du NADH et du FAD. Enfin nous détectons assez de photons pour disposer d‘informations quantitatives et donc de proposer une image du rapport d’oxydo-réduction optique en endomicroscopie. / Nonlinear microscopy is a cutting edge imaging modality leading to remarkable step forward in biology but also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there has been several development of fiber-based micro-endoscope. The application for those probes is now limited by few major restrictions, such as the impossibility to collect auto-fluorescence signal from tissues theses being inherently weak such as the fluorescence from NADH or FAD. This limitation reduces the usefulness of the micro-endoscope effectively restraining it to morphological imaging modality requiring staining of the tissue. Our aim is to go beyond this limitation, showing cellular metabolism monitoring, in real time, without any staining. The experimental setup is an upgrade of our precedent one where the reflection- based Grism stretcher is replace with a new generation transmission-based Grism stretcher. Another Laser was also added in order to tune the first laser at 860nm to allow FAD imaging and the second one to 760nm for NADH. The results prove that we assess and image the level of NADH and FAD at subcellular resolution through a five-meter-long fiber. Thus we demonstrate that we are capable of measuring the optical redox ratio in a micro-endoscopic configuration.