An on-line chromatic and scale-space microvasculature-tracing analysis for transmitted light optical images

Limited contrast in optical images from intravital microscopy is problematic for analysing tumour vascular morphology. Moreover, in some cases, changes in vasculature are visible to a human observer but are not easy to quantify. In this paper two quantitative on-line algorithms are presented: scale-space vessel tracing and chromatic decomposition for tumour vasculature from in-vivo transmitted light optical images. The algorithms were tested on intravital window chamber images of the vasculature from SW1222 human colorectal carcinomas, which were treated with a vascular disrupting agent combretastatin-A-4-phosphate (CA-4-P) or saline. The results confirmed the well-known effects of CA-4-P on the constriction of vessels. Furthermore, changes in the chromaticity suggest a deoxygenation of the blood with a recovery to initial levels in CA-4-P-treated tumours relative to the controls. The algorithms can be freely applied to any vascular image through the CAIMAN (CAncer IMage ANalysis: http://www.caiman.org.uk)

Transient absorption imaging of hemeprotein in fresh muscle fibers

2022 Summer.Includes bibliographical references.Mitochondrial diseases affect 1 in 4000 individuals in the U.S. among adults and children of all races and genders. Nevertheless, these diseases are hard to diagnose because they affect each person differently. Meanwhile the gold standard diagnosis methods are usually invasive and time- consuming. Therefore, a non-invasive and in-vivo diagnosis method is highly demanded in this area. Our goal is to develop a non-invasive diagnosis method based on the endogenous nonlinear optical effect of the live tissues. Mitochondrial disease is frequently the result of a defective electron transport chain (ETC). Our goal is to develop a non-invasive way to measure redox within the ETC, specifically, of cytochromes. Cytochromes are iron porphyrins that are essential to the ETC. Their redox states can indicate cellular oxygen consumption and mitochondrial ATP production. So being able to differentiate the redox states of cytochromes will offer us a method to characterize mitochondrial function. Meanwhile, Chergui's group found out that the two redox states of cytochrome c have different pump-probe spectroscopic responses, meaning that the transient absorption (TA) decay lifetime can be a potential molecular contrast for cytochrome redox state discrimination. Their research leads us to utilize the pump-probe spectroscopic idea to develop a time-resolved optical microscopic method to differentiate not only cytochromes from other chemical compounds but also reduced cytochromes from oxidized ones. This dissertation describes groundbreaking experiments where transient absorption is used to reveal excited-state lifetime differences between healthy controls and an animal model of mitochondrial disease, in addition to differences between reduced and oxidized ETC in isolated mitochondria and fresh preparations of muscle fibers. For our initial experiments, we built a pump-probe microscopic system with a fiber laser source, producing 530nm pump and 490nm probe using a 3.5kHz laser scanning rate. The pulse durations of pump and probe are both 800fs. For the preliminary results, we have successfully achieved TA decay contrast between reduced and oxidized cytochromes in solution form. Then we have achieved SNR enhanced pump-probe image of BGO crystal particles with the help of the software- based adaptive filter noise canceling method. We also have installed a FPGA-based adaptive filter to enhance the pump-probe signals of the electrophoresis gels that contain different mitochondrial respiratory chain supercomplexes. However, because the noise floor was still 30 dB higher than shot noise limit, cytochrome imaging in live tissues was still problematic. We then built another pump-probe microscope with a solid- state ultrafast laser source. In that way, we do not need to worry about laser relative intensity noise (RIN) anymore, since the noise floor of the solid-state laser source can reach the shot noise limit at MHz region. One other advantage of the new laser source is that it can provide one tunable laser output that can be directly converted to the probe pulse with tunable center wavelength. Its tunability can cover the entire visible spectrum. We realized a pump-probe microscopy with a 520nm pump pulse and a tunable probe pulse. The tunability on the probe arm allows us to explore better pump-probe contrast between two redox states. What's more, I will introduce my preliminary results of utilizing supercontinuum generation in a photonic crystal fiber (PCF) to realize tunability on pump wavelength. In that way, more possibilities will be unlocked. And the hyperspectral pump-probe microscope will be able to distinguish more molecules

Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy

Multiphoton microscopy is a powerful optical imaging modality renowned for its non-invasive nature and relatively affordable characteristics. In particular, it has found its niche in neuroimaging due to its ability to probe in vivo biological processes in scattering brain tissue approaching millimeter depths with cellular resolution. However, the brain is a large and complex organ, and in order to fully understand its heterogeneous architecture and associated functional roles, several distal regions must be imaged simultaneously. Moreover, due to the critical implications of organelle features in various macroscale processes, whole-brain imaging at subcellular resolution scales presents itself as one of the outstanding challenges faced by the neuroscientific community today. Primarily, this research aims to expand the depth, field-of-view, and temporal throughput of multiphoton microscopy to enable large volume imaging of microvasculature at greater acquisition speeds. To accomplish this, we combine multi-faceted efforts focused on the engineering and development of advanced multiphoton microscopy techniques and technologies. This includes the characterization of novel contrast agents, the optimization of scan system optics, and the integration of high-repetition rate lasers with a resonant galvanometer. In addition, we develop a two-color imaging system capable of enhancing excitation efficiency, improving signal-to- background ratio, and further extending imaging depth. Finally, we present a novel application for two-color non-degenerate mode mixing to effectively circumvent the diffraction-limited nature of optical resolution and enable subcellular imaging. Collectively, these efforts advance the state-of-the art of multiphoton microscopy for routine cerebrovascular and neuroimaging.Biomedical Engineerin

Cerebrovascular Dysfunction and Degeneration in Alzheimer’s Disease Pathophysiology

Alzheimer’s disease (AD) is a terminal illness and the most common form of dementia, which disproportionately affects the aged population. The pathophysiology of AD is characterized by neurodegeneration that slowly progresses, affecting regions of the brain that are involved in learning, memory, language, and executive function. In patients with the disease, early symptoms include non-disruptive forgetfulness that evolves into the inability to form new memories and ultimately the loss of autonomy at late stages. Histopathological hallmarks in the brain from patients with AD is the presence of amyloid-β (Aβ)-plaques and neurofibrillary tangles (NFT) deposited in the parenchyma. Since the discovery of these hallmarks, the majority of AD research has disproportionately focused on Aβ -plaques and NFT. Although the etiology of AD remains unknown, considerable advances have been made describing the cellular, molecular, and genetic contributions to the disease. Aging is the important risk factor for the development of AD, many other factors that increase the risk of developing AD later in life are vascular in nature. The function of the cardiovascular system is known to decline during healthy aging, and the same is true for the cerebrovasculature. Empirical evidence has demonstrated a decline cerebrovascular function in AD that exceeds the decline that occurs in healthy aging. Cerebrovascular dysfunction is the major contributor to the development of hypoperfusion and hypometabolism in patients diagnosed with AD. Cerebral amyloid angiopathy (CAA) is a neuropathological condition defined by the abnormal accumulation of Aβ on the walls of the cerebrovasculature. CAA occurs in as many as 90% of patients with AD and is implicated in the weakening of the walls of cerebral blood vessels. The occurrence of microhemorrhages, aneurysms, and microinfarctions are pathological manifestations associated with weakened walls of cerebral blood vessels in the brains of patients with confirmed AD. Noteworthy, cerebrovascular dysfunction, hypoperfusion, and hypometabolism occur before the onset of Aβ-plaque and NFT deposition in the brain of patients and animal models with AD. These findings provide a compelling basis that suggest a prominent role of dysfunctional cerebrovasculature in the etiology and for the progression of AD. Although the overwhelming evidence that implicates cerebrovascular dysfunction in AD, a thorough account of the changes that occur to the cerebrovasculature nor the mechanisms that drive these changes during the development and progression of AD has not been previously reported. The overarching goal(s) of this work are to; (1) provide a thorough description of the changes that occur to the cerebrovasculature during age and the progression of AD; (2) describe the mechanisms involved in cerebrovascular damage in AD; and (3) characterize the degeneration that results from cerebrovascular hypoperfusion. These overarching goals were achieved by completing five separate studies. Described in study 1, we investigated the effects of hypoxia on astrocytic mitochondria by assessing mitochondrial fission-fusion dynamics, reactive oxygen species production, synthesis of ATP, and mitophagy. Overall, we found a drastic mitochondrial network change that is triggered by metabolic crisis during hypoxia; these changes are followed by mitochondrial degradation and retraction of astrocytic extensions during reoxygenation. In study 2, we provide a novel model for the gradual development of cerebrovascular hypoperfusion in mice. Cerebrovascular hypoperfusion developed over 34-days by inserting an ameroid constrictor ring and microcoil bilaterally around the external carotid arteries. We investigated the neurodegenerative effects of hypoperfusion in mice by assessing both gray and white matter pathology. Histopathological analyses of the brain revealed neuronal and axonal degeneration as well as necrotic lesions. The most severely affected regions were located in the hippocampus and corpus callosum. Described in study 3, we performed a series of experiments to investigate the effects of Aβ on cerebrovascular endothelial cells. In this study, we focused on characterizing the changes to mitochondrial oxidative phosphorylation, superoxide production, mitochondrial calcium, ATP synthesis, and endothelial cell death. These results describe a mechanism for mitochondrial degeneration caused by the production of mitochondrial superoxide, which was driven by increased mitochondrial Ca2+ uptake. We found that persistent superoxide production injures mitochondria and disrupts electron transport in cerebrovascular endothelial cells. In study 4, we developed a method to evaluate the cerebrovasculature of the whole-brain and constructed analyses to assess the angioarchitecture. We used vascular corrosion casting method to replicate the cerebrovasculature in adult mice and used MicroCT to acquire volumetric imaging data of the cerebrovascular network at a resolution required to investigate the microvasculature. Our analyses of the cerebrovasculature evaluated the morphology, topology, and organization of the angioarchitecture. With these developments, we investigated the effects of age and progression of disease on the cerebrovasculature in wild type mice and the triple transgenic mouse model of AD. Study 5 provides data describing degenerative changes to the microvascular network that progress with age in the triple transgenic mouse model of AD. These changes to the microvasculature occurred early, before the onset of Aβ-plaque deposition and NFT development. Overall, this body of work provides evidence of an early cerebrovascular disruption in the etiology of AD that progresses with age. Aβ mediates early cerebrovascular damage through direct interaction with vascular endothelial cells. Microvascular degeneration can lead to hypoperfusion which damages both gray and white matter. Hypoperfusion-associated hypoxia may mediate parenchymal damage by disrupting mitochondrial fission-fusion dynamics and enhancing mitophagy. These data provide a basis for the development of novel therapeutic strategies that target the changes to the cerebrovasculature for the treatment of AD. These observations may substantiate a prophylactic strategy for the treatment of AD by preventing the initial factors that lead to compromised cerebrovasculature

New MRI Techniques for Nanoparticle Based Functional and Molecular Imaging

Although in clinical use for several decades, magnetic resonance imaging: MRI) is undergoing a transition from a qualitative anatomical imaging tool to a quantitative technique for evaluating myriad diseases. Furthermore, MRI has made great strides as a potential tool for molecular imaging of cellular and tissue biomarkers. Of the candidate contrast agents for molecular MRI, the excellent bio-compatibility and adaptability of perfluorocarbon nanoparticles: PFC NP) has established these agents as a potent targeted imaging agent and as a functional platform for non-invasive oxygen tension sensing. Direct readout and quantification of PFC NP can be achieved with fluorine: 19F) MRI because of the unique 19F signal emanating from the core PFC molecules. However, the signal is usually limited by the modest accumulated concentrations as well as several special NMR considerations for PFC NP, which renders 19F MRI technically challenging in terms of detection sensitivity, scan time, and image reconstruction. In the present dissertation, some of the pertinent NMR properties of PFC NP are investigated and new 19F MRI techniques developed to enhance their performance and expand the biomedical applications of 19F MRI with PFC NP. With the use of both theoretical and experimental methods, we evaluated J-coupling modulation, chemical shift and paramagnetic relaxation enhancement of PFC molecules in PFC NP. Our unique contribution to the technical improvement of 19F MRI of small animal involves:: 1) development of general strategies for RF 1H/19F coil design;: 2) design of novel MR pulse sequences for 19F T1 quantification; and: 3) optimization of imaging protocols for distinguishing and visualizing multiple PFC components: multi-chromatic 19F MRI). The first pre-clinical application of our novel 19F MRI techniques is blood vessel imaging and rapid blood oxygen tension measurement in vivo. Blood vessel anatomy and blood oxygen tension provide pivotal physiological information for routine diagnosis of cardiovascular disease. Using our novel Blood: flow)-Enhanced-Saturation-Recovery: BESR) sequence, we successfully visualized reduced flow caused by thrombosis in carotid arteries and jugular veins, and we quantified the oxygen tension in the cardiac ventricles of the mouse. The BESR sequence depicted the expected oxygenation difference between arterial and venous blood and accurately registered the response of blood oxygen tension to high oxygen concentration in 100% oxygen gas. This study demonstrated the potential application of PFC NP as a blood oxygen tension sensor and blood pool MR contrast agent for angiography. Another pre-clinical application investigated was functional kidney imaging with 19F MRI of circulating PFC NP. Conventional functional kidney imaging typically calls for the injection of small molecule contrast agents that may be nephrotoxic, which raises concerns for their clinical applications in patients with renal insufficiency. We demonstrated that our 19F MRI technique offers a promising alternative functional renal imaging approach that generates quantitative measurement of renal blood volume and intrarenal oxygenation. We successfully mapped the expected heterogeneous distribution of renal blood volume and confirmed the presence of an oxygenation gradient in healthy kidneys. We validated the diagnostic capability of 19F MRI in a mouse model of acute ischemia/reperfusion kidney injury. We also employed 19F MRI as a tool to test the therapeutic efficacy of a new nanoparticle-based drug, i. e. PPACK: D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone) PFC NP, which was postulated to inhibit microvascular coagulation during acute kidney injury. Based on our preliminary 19F MRI findings, we observed that PPACK PFC NP effectively reduced coagulation in our animal model, as evidenced by lesser accumulation of particles trapped by the clotting process. This finding suggests the potential for 19F MRI to be used as a drug monitoring tool as well in common medical emergencies such as acute kidney failure

Human retinal oximetry using hyperspectral imaging

The aim of the work reported in this thesis was to investigate the possibility of measuring human retinal oxygen saturation using hyperspectral imaging. A direct non-invasive quantitative mapping of retinal oxygen saturation is enabled by hyperspectral imaging whereby the absorption spectra of oxygenated and deoxygenated haemoglobin are recorded and analysed. Implementation of spectral retinal imaging thus requires ophthalmic instrumentation capable of efficiently recording the requisite spectral data cube. For this purpose, a spectral retinal imager was developed for the first time by integrating a liquid crystal tuneable filter into the illumination system of a conventional fundus camera to enable the recording of narrow-band spectral images in time sequence from 400nm to 700nm. Postprocessing algorithms were developed to enable accurate exploitation of spectral retinal images and overcome the confounding problems associated with this technique due to the erratic eye motion and illumination variation. Several algorithms were developed to provide semi-quantitative and quantitative oxygen saturation measurements. Accurate quantitative measurements necessitated an optical model of light propagation into the retina that takes into account the absorption and scattering of light by red blood cells. To validate the oxygen saturation measurements and algorithms, a model eye was constructed and measurements were compared with gold-standard measurements obtained by a Co-Oximeter. The accuracy of the oxygen saturation measurements was (3.31%± 2.19) for oxygenated blood samples. Clinical trials from healthy and diseased subjects were analysed and oxygen saturation measurements were compared to establish a merit of certain retinal diseases. Oxygen saturation measurements were in agreement with clinician expectations in both veins (48%±9) and arteries (96%±5). We also present in this thesis the development of novel clinical instrument based on IRIS to perform retinal oximetry.Al-baath University, Syri