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
