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

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

In Vivo Molecular Targeted Imaging of Cancer

Hepatocellular carcinoma (HCC) presents a global healthcare problem. It is the second most lethal cancer worldwide, causing 745,000 deaths annually. HCC accounts for 70% to 90% of primary liver cancer cases with rising incidence in developed countries. Newly diagnosed cases in the U.S. are expected to increase by 10% in three years. Symptoms of HCC typically do not appear until advanced stage, leaving surgical resection the primary therapy. However, HCC patients suffer from dire prognosis of less than 5% five-year survival rate and >50% incidence of tumor recurrence, due to poor contrast of HCC against surrounding liver tissue limiting resection accuracy. Using a molecular imaging system that targets differentially expressed tumor specific surface biomarkers may help detect HCC neoplasm missed by surgeons and preserve viable liver tissue to reduce recurrence and improve patient recovery. This dissertation presents the HCC targeting and imaging methods developed to specifically identify HCC neoplasm with high contrast, fast kinetics and deep penetration. Two cancer cell surface biomarkers, epidermal growth factor receptor (EGFR) and glypican-3 (GPC3), are important in the development of HCC. To create a molecular imaging strategy for HCC detection, short peptide sequences specifically binding to these biomarkers have been selected and validated. They demonstrated high target affinities (kd < 75 nM) and fast cellular binding kinetics (<10 min). After conjugating with near-infrared organic dye, these molecular targeting probes were able to home to the HCC tumor xenograft in vivo after intravenous administration. Ex vivo and in vivo optical imaging was conducted with fluorescent laparoscopy, whole body fluorescent imaging, and hand held dual-axis confocal microscopy. In vivo cell surface binding of peptide probe to HCC xenograft in mice was observed at subcellular resolution in both horizontal (1000×1000 µm2) and vertical (1000×430 µm2) planes. Tumor margins were automatically detected with computerized segmentation algorithm. High target-to-background ratios (2.99 and 6.2 respectively) were achieved at tumor sites after 6 and 2 hours respectively, and targeting probes were cleared from the animal system within 24 hours. In addition, targeted in vivo photoacoustic tomography (PAT) imaging visualized probe penetration inside the tumor 1.8 cm beneath intact skin. Plasmonic nanoparticles absorb light more efficiently than organic dyes. By coating GPC3 targeting peptide onto gold nanoshell (GNS) surface, in vivo photoacoustic imaging contrast was improved from 2.25 to 4.45 and imaging depth reached 2.1 cm. Peak probe uptake in vivo occurred at 2 hours and clearance took place within 12 hours, which are desirable pharmacokinetics for clinical settings of intraoperative imaging guidance. Specific binding, biodistribution and toxicity were investigated in cultured cells, ex vivo tissues (human and mouse) as well as in mouse models. The GPC3 targeting probe was able to distinguish HCC from non-HCC human patient biopsies (n=41) at 93% sensitivity and 88% specificity, with area under the receiver operator characteristic curve (AUC) value reaching 0.98. These studies showed that affinity peptide based molecular imaging is an enabling technology which will allow clinicians to perform functional imaging during surgery to identify resection margin with high contrast, sensitivity and speed.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138522/1/zhouquan_1.pd

XXIV congreso anual de la sociedad española de ingeniería biomédica (CASEIB2016)

En la presente edición, más de 150 trabajos de alto nivel científico van a ser presentados en 18 sesiones paralelas y 3 sesiones de póster, que se centrarán en áreas relevantes de la Ingeniería Biomédica. Entre las sesiones paralelas se pueden destacar la sesión plenaria Premio José María Ferrero Corral y la sesión de Competición de alumnos de Grado en Ingeniería Biomédica, con la participación de 16 alumnos de los Grados en Ingeniería Biomédica a nivel nacional. El programa científico se complementa con dos ponencias invitadas de científicos reconocidos internacionalmente, dos mesas redondas con una importante participación de sociedades científicas médicas y de profesionales de la industria de tecnología médica, y dos actos sociales que permitirán a los participantes acercarse a la historia y cultura valenciana. Por primera vez, en colaboración con FENIN, seJane Campos, R. (2017). XXIV congreso anual de la sociedad española de ingeniería biomédica (CASEIB2016). Editorial Universitat Politècnica de València. http://hdl.handle.net/10251/79277EDITORIA