MEMS-Based Endomicroscopes for High Resolution in vivo Imaging

Intravital microscopy is an emerging methodology for performing real time imaging in live animals. This technology is playing a greater role in the study of cellular and molecular biology because in vitro systems cannot adequately recapitulate the microenvironment of living tissues and systems. Conventional intravital microscopes use large, bulky objectives that require wide surgical exposure to image internal organs and result in terminal experiments. If these instruments can be reduced sufficiently in size, biological phenomena can be observed in a longitudinal fashion without animal sacrifice. The epithelium is a thin layer of tissue in hollow organs, and is the origin of many types of human diseases. In vivo assessment of biomarkers expressed in the epithelium in animal models can provide valuable information of disease development and drug efficacy. The overall goal of this work is to develop miniature imaging instruments capable of visualizing the epithelium in live animals with subcellular resolution. The dissertation is divided into four projects, where each contains an imaging system developed for small animal imaging. These systems are all designed using laser beam scanning technology with tiny mirrors developed with microelectromechanical systems (MEMS) technology. By using these miniature scanners, we are able to develop endomicroscopes small enough for hollow organs in small animals. The performance of these systems has been demonstrated by imaging either excised tissue or colon of live mice. The final version of the instrument can collect horizontal/oblique plane images in the mouse colon in real time (>10 frames/sec) with sub-micron resolution (<1 um), deep tissue penetration (~200 um) and large field of view (700 x 500 um). A novel side-viewing architecture with distal MEMS scanning was developed to create clear and stable image in the mouse colon. With the use of the instrument, it is convenient to pinpoint location of interest and create a map of the colon using image mosaicking. Multispectral fluorescence images can by collected at excitation wavelength ranging from 445 nm to 780 nm. The instruments have been used to 1) validate specific binding of a cancer targeting agent in the mouse colon and 2) study the tumor development in a mouse model with endogenous fluorescence protein expression. We use these studies to show that we have developed an enabling technology which will allow biologist to perform longitudinal imaging in animal models with subcellular resolution.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/136954/2/dxy_1.pd

Multimodal Optical Imaging by Microendoscope

In the past decades, optical imaging field has been developing rapidly. Noninvasive imaging enabled by microendoscopes has become a promising tool for early cancer detection and imaging-guided surgery. In this chapter, we will mainly introduce most advances in the miniaturized microendoscope development, including photoacoustic, confocal fluorescence, multiphoton fluorescence, second-harmonic generation (SHG) label-free imaging, wide-field fluorescence, surface-enhanced Raman scattering (SERS) nanoparticle-based Raman spectroscopy. Enabled by the frontier micromachining techniques, micro-opto-electromechanical system (MOEMS)-based novel microendoscopes with various imaging modalities have been prototyped and further translated into clinics. The working principle of representative microendoscopes and optical imaging modalities will be introduced in detail

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

A Dual-modality Smartphone Microendoscope for Quantifying the Physiological and Morphological Properties of Epithelial Tissues

We report a nonconcurrent dual-modality fiber-optic microendoscope (named SmartME) that integrates quantitative diffuse reflectance spectroscopy (DRS) and high-resolution fluorescence imaging (FLI) into a smartphone platform. The FLI module has a spatial resolution of ~3.5 µm, which allows the determination of the nuclear-cytoplasmic ratio (N/C) of epithelial tissues. The DRS has a spectral resolution of ~2 nm and can measure the total hemoglobin concentration (THC) and scattering properties of epithelial tissues with mean errors of 4.7% and 6.9%, respectively, which are comparable to the errors achieved with a benchtop spectrometer. Our preliminary in vivo studies from a single healthy human subject demonstrate that the SmartME can noninvasively quantify the tissue parameters of normal human oral mucosa tissues, including labial mucosa tissue, gingival tissue, and tongue dorsum tissue. The THCs of the three oral mucosa tissues are significantly different from each other (p ≤ 0.003). The reduced scattering coefficients of the gingival and labial tissues are significantly different from those of the tongue dorsum tissue (p \u3c 0.001) but are not significantly different from each other. The N/Cs for all three tissue types are similar. The SmartME has great potential to be used as a portable, cost-effective, and globally connected tool to quantify the THC and scattering properties of tissues in vivo

Development of a single-mode interstitial rotary probe for In Vivo deep brain fluorescence imaging

Ce mémoire rend compte de l'expertise développée par l'auteur au Centre de recherchede l'Institut universitaire en santé mentale de Québec (CRIUSMQ) en endoscopie fibrée. Il décrit la construction d'un nouveau type de microscope optique, le MicroscopeInterstitiel Panoramique (PIM). Par la juxtaposition d'un court morceau de fibre à gradientd'indice et d'un prisme à l'extrémité d'une fibre monomode, la lumière laser estfocalisée sur le côté de la sonde. Pour former une image, cette dernière est rapidementtournée autour de son axe pendant qu'elle est tirée verticalement par un actuateurpiézo-électrique. Ce design de système rotatif d'imagerie interstitielle peu invasif est uneffort pour limiter les dégâts causés par la sonde tout en imageant la plus grande régionpossible en imagerie optique cérébrale profonde.This thesis documents the expertise developed by the author at the Centre de recherchede l'Institut universitaire en santé mentale de Québec (CRIUSMQ) in fibered endoscopy, particularly the design and construction of a new kind of optical microscope: ThePanoramic Interstitial Microscope (PIM). Through the juxtaposition of a short piece ofGraded-Index fibre and a prism at the end of a single-mode fibre, laser light is focussedon the side of the probe. To form an image, the latter is quickly spun around its axiswhile it is being pulled vertically by a piezoelectric actuator. This minimally invasivefluorescence rotary interstitial imaging system is an endeavor to limit the damage causedby the probe while imaging enough tissue to provide good context to the user in deep brain optical imaging

Development of a multimodal foveated endomicroscope for the detection of oral cancer

A multimodal endomicroscope was developed for cancer detection that combines hyperspectral and confocal imaging through a single foveated objective and a vibrating optical fiber bundle. Standard clinical examination has a limited ability to identify early stage oral cancer. Optical detection methods are typically restricted by either achievable resolution or a small field-of-view. By combining high resolution and widefield spectral imaging into a single probe, a device was developed that provides spectral and spatial information over a 5 mm field to locate suspicious lesions that can then be inspected in high resolution mode. The device was evaluated on ex vivo biopsies of human oral tumors

Achromatized endomicroscope objective for optical biopsy

Currently, researchers and clinicians lack achromatized endomicroscope objectives that are as narrow as biopsy needles. We present a proof-of-concept prototype that validates the optical design of an NA0.4 objective. The objective, built with plastic lenses, has a 0.9 mm clear aperture and is achromatized from 452 nm to 623 nm. The objective’s measured Strehl ratio is 0.74 ± 0.05 across a 250 μm FOV. We perform optical sectioning via structured illumination through the objective while capturing fluorescence images of breast carcinoma cells stained with proflavine and cresyl violet. This technology has the potential to improve optical biopsies and provide the next step forward in cancer diagnostics