MULTI-MODAL OPTICAL NEUROIMAGING AND APPLICATIONS

Optical imaging tools provide superior details than MRI, PET in monitoring the physiological and pathological state of brain in preclinical models. By combining different optical imaging modalities, a variety of physiological parameters (e.g. cerebral hemodynamics, metabolism and neuronal activity) could be detected simultaneously; such simultaneous imaging is expected to profoundly enhance our understanding of normal brain regulation and its disruption from neurovascular disorders. As a part of this thesis, I designed a multi-modal optical imaging system that could perform simultaneous laser speckle contrast imaging, wide-field fluorescence imaging and optical intrinsic signal imaging. The principles, processing methods and applications of these imaging modalities are presented in Chapter 1. The system was first applied to study a new atherothrombotic stroke model and to evaluate the recovery of stroke from different treatment protocols in mice (Chapter 2). Cerebral blood flow changes and thrombus formations were imaged by laser speckle contrast imaging and wide-field fluorescence imaging, respectively. We concluded that the combination treatment of tissue plasminogen activator and cathepsin G inhibitor improved the neurological outcomes of ischemic brain injury from induced atherothrombotic stroke. To investigate brain activity in high-resolution by optical imaging tools, cranial window preparation is an essential procedure to allow optical access to the brain. We also employed the optical imaging system to investigate the effects of cranial windows on monitoring neurovasculature by laser speckle contrast imaging (Chapter 3). Open-skull and thin-skull cranial window procedures were performed in separate experiments, and the neurovasculature underlying the cranial windows were monitored for fourteen days. The differences between two window types were systematically compared by parameters such as contrast-to-noise ratio and microvessel density. Finally, the last part of my thesis was to miniaturize the multi-modal bench-top imaging system to a head-mounted microscope, which allows imaging on awake freely moving animals. The natural physiological state of brain activities can be detected without the confounding effects of anesthetics. The current version of the microscope weighs less than 5 g and is able to perform laser speckle contrast imaging, wide-field fluorescence imaging and optical intrinsic signal imaging simultaneously. We are currently testing the miniaturized microscope to study a brain tumor murine model. Finally, I describe the current progress of miniaturized optical neuroimaging systems on awake moving animals in Chapter 4 of this thesis

DEVELOPEMENT OF WIDEFIELD MULTI-CONTRAST OPTICAL METHODS FOR IN VIVO MICROVASCULAR SCALE IMAGING

Traditional in vivo optical imaging methods rely on a single contrast mechanism, thereby limiting one’s ability to characterize more than one biological variable. However, most biological systems are complex and are comprised of multiple variables. Therefore, optical methods that employ multiple contrast mechanisms and are capable of visualizing multiple biological variables would permit a more comprehensive understanding of biological systems. Multi-contrast optical imaging, therefore, has great potential for both fundamental and applied biomedical research. The goal of this dissertation is to develop optical methods to enable multi-contrast imaging in vivo over a wide field of view while retaining a microvascular scale spatial resolution. We present the integration of three types of optical imaging contrast mechanisms: fluorescence (FL), intrinsic optical signals (IOS) and laser speckle contrast (LSC). Fluorescence enables tracking pre-labelled molecules and cells, IOS allow quantification of blood volume and/or intravascular oxygen saturation, and LSC permits assessment of tissue perfusion. Together, these contrast mechanisms can be harnessed to provide a more complete picture of the underlying physiology at the microvascular spatial scale. We developed two such microvascular resolution optical multi-contrast imaging methods, and demonstrated their utility in multiple biomedical applications. First, we developed a multi-contrast imaging system that can interrogate in vivo both neural activity and its corresponding microvascular scale hemodynamics in the brain of a freely moving rodent. To do this, we miniaturized an entire benchtop optical imaging system that would typically occupy 5 x 5 x 5 feet, into just 5 cm3. Our miniaturized microscope weighs only 9 g. The miniature size and light weight permitted us to mount our microscope on a rodent’s head and image brain activity in vivo with multiple contrast mechanisms. We used our microscope to study the functional activation of the mouse auditory cortex, and to investigate the alteration of brain function during arousal from deep anesthesia. Our miniaturized microscope is the world’s first rodent head-mountable imaging system capable of interrogating both neural and hemodynamic brain activity. We envision our microscope to usher an exciting new era in neuroscience research. Second, we developed an optical imaging system to extensively characterize microvascular scale hemodynamics in vivo in an orthotopic breast tumor model. We specifically designed it as a benchtop based system to allow ample space for surgical preparation and small animal manipulation. Using it, we continuously monitored in vivo microvascular scale changes in tissue perfusion, blood volume and intravascular oxygen saturation of an orthotopic breast tumor microenvironment for multiple hours over a field of view encompassing the entire tumor extent. This unique dataset enabled us for the first time to characterize the temporal relationship between different tumor hemodynamic variables at the scale of individual microvessels. We envision our work to inspire a whole new avenue of experimental cancer research where the role of a tumor’s hemodynamic microenvironment is extensively characterized at its native (i.e. microvascular) spatial scale. In summary, this dissertation describes the design, implementation and demonstration of two microvascular resolution, wide-field, multi-contrast optical imaging systems. We believe these methods to be a new tool for broadening our understanding of biology