Microelectronic vision prostheses aim to restore visual percepts through electrical stimulation of the surviving visual pathways in the blind. Electrical stimulation has been shown to produce spots of light in the visual field. A neurostimulator that forms the basis of a vision prosthesis was designed using a high voltage CMOS process to allow it to be able to stimulate when faced with high electrode-tissue impedances. It was implemented with novel features that allow it to be scalable, and to focus charge injection, and can stimulate multiple sites simultaneously using a current source and sink at each site. To reduce electrical cross-talk between multiple stimulation sites, six-return electrodes surround each stimulating electrode, electrically guarding them from each other. The six-return electrode configuration was shown to reduce electrical cross-talk in saline bath tests compared to single-return electrode configurations. The neurostimulator was used to evoke responses from cats through electrical stimulation via intravitreal ball electrodes, corneal electrodes, and planar electrode arrays in the suprachoroidal space. Responses were measured on the visual cortex through optical imaging of intrinsic signals, and through surface electrodes. Using the planar electrode array in the suprachoroidal space, responses were elicited to biphasic, bipolar and monopolar stimuli, with each stimulating electrode coupled with either six-return electrodes, two-return electrodes, or a single-return electrode. The average charge threshold to elicit a response for biphasic, bipolar stimulation with six-return electrodes was 76.47 ?? 8.76 nC (standard error of the mean). For biphasic, bipolar stimulation, the magnitude and area of cortical response with the six-return electrode configurations was on average 2.18 ?? 0.19 times smaller than single-return electrode configurations, and 1.89 ?? 0.19 times smaller than two-return electrode configurations (P < 0.0001). It was also found that for biphasic stimulation, a greater magnitude and area of response was elicited for monopolar stimulation compared to bipolar stimulation. This dissertation details the design and testing of a novel, scalable neurostimulator to focus charge injection. It also shows that suprachoroidal, bipolar stimulation can elicit visual responses, and that the area of cortical activation was more focused when using bipolar, biphasic stimulation, and six-return electrodes.

Neuroinflammation is a major driver of secondary brain cell death after ischaemic stroke, seizure activity and traumatic brain injury. In a model of excitotoxic neuroinflammation, striatal injection of a toxic dose of AMPA causes cell death in the striatum after 24 hours. Co-injection of AMPA with the pro-inflammatory cytokine interleukin-1β (IL-1β) leads to additional cortical cell death. Injected alone, IL-1β leads to little or no cell death. It is hypothesised that IL-1β may exacerbate cell death by interfering with blood flow coupling. In the first study, two-dimensional optical imaging spectroscopy was used to measure early changes in the haemodynamic response in the anaesthetised rat barrel cortex before and for 6 hours after injection of vehicle, AMPA, IL-1β, or AMPA+IL-1β. After injection of IL-1β, with or without AMPA, the oxygenated blood flow response to mechanical whisker stimulation approximately halved over the course of 6h. In the second study, to determine whether the IL-1β-dependent changes in blood flow response are reflected by altered cellular activity, local field potentials, multi-unit activity and local tissue oxygenation responses to whisker stimulation were recorded simultaneously from the active barrel before and up to 6h after injection. A similar reduction in the size of the oxygenation response was seen again in the IL-1β- and AMPA+IL-1β-treated groups. Importantly, the level of gamma frequency oscillations at stimulus onset decreased within the first hours after injection of AMPA+IL-1β or IL-1β, suggesting a disruption of the fast-spiking interneuron network in the barrel cortex. These findings, along with histological observations of IL-1β-dependent markers of neuroinflammation, suggest that IL-1β may exacerbate AMPA-induced excitotoxicity by potentiating seizure activity and decoupling the neurovascular response in the cortex.

Pulmonary fibrosis is a chronic and debilitating condition that proposes several challenges to both veterinary and medical clinicians. Despite considerable research, many fibrotic lung diseases remain elusive in terms of aetiology, pathogenesis and treatment. Furthermore, progress is hindered by the lack of a translatable animal model with durable and persistent fibrosis. Asinine Pulmonary Fibrosis (APF) is a spontaneous syndrome of aged donkeys with high prevalence (35%). No previous detailed characterisation of APF has been performed and disease diagnosis remains a challenge. APF was studied with regard to clinical, pathological and molecular features and the suitability of this condition as a model for a rare fibrotic lung disease in humans known as pleuroparenchymal fibroelastosis (PPFE) was assessed. In addition, target activatable optical imaging reagents for the real time detection of two key molecular markers of fibrosis: matrix metalloproteinases (MMPs) and lysyl oxidases (LOXF) were evaluated in spontaneous ex vivo models of fibrosis. Such reagents may be used alongside fibred confocal fluorescence microscopy (FCFM), a relatively noninvasive and cutting edge diagnostic tool, to detect and monitor fibroproliferation in animals and man. Whole lungs were collected from 32 aged donkeys at routine necropsy. Gross examination revealed pulmonary fibrosis in 19 donkeys (APF cases), while 13 (controls) had grossly normal lungs. HRCT images and histology sections were reviewed independently and blindly for each of the lungs. Ten of 19 APF lungs were categorised as being ‘consistent with’ PPFE according to previously defined histological and imaging criteria. All 10 PPFE-like lungs had marked pleural and subpleural fibrosis, predominantly within the upper lung zone, with accompanying intra-alveolar fibrosis and elastosis. An activatable Smartprobe for the detection of LOXF, TWB-219, was synthesised by The Bradley Group, Department of Chemistry (UoEDC). The probe was based on a tandem amine oxidation and β-elimination mechanism, resulting in signal amplification detected at the 488nm wavelength. The probe showed increased fluorescence in the presence of diamine oxidase as well as on incubation with aged human lung tissue cell-free homogenate as determined by a fluorescent plate reader. This signal amplification could be inhibited by β-aminopropionitrile, a recognised LOX inhibitor as well as by an in-house inhibitor specific to LOX. An evolutionary family of MMP probes with varying cleavage sequences and structures, synthesised by the UoEDC, was evaluated at each stage of progression with regard to signal to noise ratio, sensitivity and specificity. Probes were tested against recombinant enzymes from the MMP family as well as neutrophil elastase and plasmin. Signal amplification was also assessed on incubation with human and ovine ex vivo lung tissue. The final ‘lead’ MMP probe, SVC-186, was cleaved by MMP-2, -9 and -13. Signal amplification was also seen following incubation with both human and ovine tissue with significant inhibition in the presence of the pan- MMP inhibitor, marimastat. In conclusion, APF is an emerging condition of aged donkeys that shares key pathological and imaging features with human PPFE. Diagnosis of APF and other fibrotic lung conditions across species remains a challenge to veterinary and medical professionals. As such, optical imaging tools may provide dynamic, real time information on the presence and progression of fibroproliferation in the lung. TWB- 219 and SVC-186 produce a detectable increase in fluorescent signal at the 488nm wavelength when activated by LOXF and MMPs respectively. These probes have been shown to function in human ex vivo tissue as assessed by FCFM.

The use of non-invasive methods to visualise and monitor processes inside living organisms is vital in the understanding and diagnosis of disease. The work in this thesis details the synthesis and applications of a new imaging modality; carbon dots, whose inherent fluorescence and non-toxic nature makes them attractive alternatives to more traditional ‘quantum dots’. In this thesis, different methods of carbon dot synthesis were attempted in order to produce carbon dots of the desired size and morphology. Nitrogen-containing carbon dots generated from 1,4-addition polymers provided the most successful route with optical and structural characteristics studied by TEM, UV-Vis and fluorescence spectroscopy and XPS. The biological behaviour of the carbon dots produced by this method were also evaluated. The ability of these carbon dots to up-convert when excited at long excitation wavelengths was studied. A number of biologically relevant applications of the carbon dots were studied. Using amine-functionalised carbon dots, cell targeting cargoes were conjugated and the effects of the carbon dot-cargo conjugates on cell lines were studied.

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.

More and more researchers and clinicians are looking to molecular sensing to predict how cells will behave, seeking the answers to questions like "will these tumor cells become malignant?" or "how will these cells respond to chemotherapy?" Optical methods are attractive for answering these questions because optical radiation is safer and less expensive than alternative methods, such as CT which uses X-ray radiation, PET/SPECT which use gamma radiation, or MRI which is expensive and only available in a hospital setting. In this dissertation, three distinct optical methods are explored to detect at the molecular level: optical coherence tomography (OCT), laser-induced fluorescence (LIF), and optical polarimetry. OCT has the capability to simultaneously capture anatomical information as well as molecular information using targeted contrast agents such as gold nanoshells. LIF is less useful for capturing anatomical information, but it can achieve significantly better molecular sensitivity with the use of targeted fluorescent dyes. Optical polarimetry has potential to detect the concentration of helical molecules, such as glucose. All of these methods are noninvasive or minimally invasive.The work is organized into four specific aims. The first is the design and implementation of a fast, high resolution, endoscopic OCT system to facilitate minimally invasive mouse colon imaging. The second aim is to demonstrate the utility of this system for automatically identifying tumor lesions based on tissue microstructure. The third is to demonstrate the use of contrast agents to detect molecular expression using OCT and LIF. The last aim is to demonstrate a new method based on optical polarimetry for noninvasive glucose sensing.

Diffuse optical tomography uses near infrared (NIR) light as the probing media to recover the distributions of tissue optical properties. It has a potential to become an adjunct imaging modality for breast and brain imaging, that is capable of providing functional information of the tissue under investigation. As NIR light propagation in the tissue is dominated by scattering, the image reconstruction problem (inverse problem) tends to be non-linear and ill-posed, requiring usage of advanced computational methods to compensate this. Traditional image reconstruction methods in diffuse optical tomography employ l2 –norm based regularization, which is known to remove high frequency noises in the re-constructed images and make them appear smooth. The recovered contrast in the reconstructed image in these type of methods are typically dependent on the iterative nature of the method employed, in which the non-linear iterative technique is known to perform better in comparison to linear techniques. The usage of non-linear iterative techniques in the real-time, especially in dynamical imaging, becomes prohibitive due to the computational complexity associated with them. In the rapid dynamic diffuse optical imaging, assumption of a linear dependency in the solutions between successive frames results in a linear inverse problem. This new frame work along with the l1–norm based regularization can provide better robustness to noise and results in a better contrast recovery compared to conventional l2 –based techniques. Moreover, it is shown that the proposed l1-based technique is computationally efficient compared to its counterpart(l2 –based one). The proposed framework requires a reasonably close estimate of the actual solution for the initial frame and any suboptimal estimate leads to erroneous reconstruction results for the subsequent frames. Modern diffuse optical imaging systems are multi-modal in nature, where diffuse optical imaging is combined with traditional imaging modalities such as MRI, CT, and Ultrasound. A novel approach that can more effectively use the structural information provided by the traditional imaging modalities in these scenarios is introduced, which is based on prior image constrained- l1 minimization scheme. This method has been motivated by the recent progress in the sparse image reconstruction techniques. It is shown that the- l1 based frame work is more effective in terms of localizing the tumor region and recovering the optical property values both in numerical and gelatin phantom cases compared to the traditional methods that use structural information.

The Optical Imaging Laboratory has developed a hand-held optical imaging system that is capable of 3D tomographic imaging. However, the imaging system is limited by longer imaging times, and inaccuracy in the positional tracking of the hand-held probe. Hence, the objective is to improve the performance of the imaging system by improving imaging time and positional accuracy. This involves: (i) development of automated single Labview-based software towards near real-time imaging; and (ii) implementation of an alternative positional tracking device (optical) towards improved positional accuracy during imaging. Experimental studies were performed using cubical tissue phantoms (1% Liposyn solution) and 0.45-cc fluorescence target(s) placed under various conditions. The studies demonstrated a 90% reduction in the imaging time (now ~27 sec/image) and also an increase from 94% to 97% in the positional accuracy of the hand-held probe. Performance enhancements in the hand-held optical imaging system have improved its potential towards clinical breast imaging.

Imaging of multiple cells has rapidly multiplied the rate of data acquisition as well as our knowledge of the complex dynamics within the mammalian brain. The process of data acquisition has been dramatically enhanced with highly affordable, sensitive image sensors enable high-throughput detection of neural activity in intact animals. Genetically encoded calcium sensors deliver a substantial boost in signal strength and in combination with equally critical advances in the size, speed, and sensitivity of image sensors available in scientific cameras enables high-throughput detection of neural activity in behaving animals using traditional wide-field fluorescence microscopy. However, the tremendous increase in data flow presents challenges to processing, analysis, and storage of captured video, and prompts a reexamination of traditional routines used to process data in neuroscience and now demand improvements in both our hardware and software applications for processing, analyzing, and storing captured video. This project demonstrates the ease with which a dependable and affordable wide-field fluorescence imaging system can be assembled and integrated with behavior control and monitoring system such as found in a typical neuroscience laboratory. An Open-source MATLAB toolbox is employed to efficiently analyze and visualize large imaging data sets in a manner that is both interactive and fully automated. This software package provides a library of image pre-processing routines optimized for batch-processing of continuous functional fluorescence video, and additionally automates a fast unsupervised ROI detection and signal extraction routine. Further, an extension of this toolbox that uses GPU programming to process streaming video, enabling the identification, segmentation and extraction of neural activity signals on-line is described in which specific algorithms improve signal specificity and image quality at the single cell level in a behaving animal. This project describes the strategic ingredients for transforming a large bulk flow of raw continuous video into proportionally informative images and knowledge.

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