Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis

Animal models of stroke are used extensively to study the mechanisms involved in the acute and chronic phases of recovery following stroke. A translatable animal model that closely mimics the mechanisms of a human stroke is essential in understanding recovery processes as well as developing therapies that improve functional outcomes. We describe a photothrombosis stroke model that is capable of targeting a single distal pial branch of the middle cerebral artery with minimal damage to the surrounding parenchyma in awake head-fixed mice. Mice are implanted with chronic cranial windows above one hemisphere of the brain that allow optical access to study recovery mechanisms for over a month following occlusion. Additionally, we study the effect of laser spot size used for occlusion and demonstrate that a spot size with small axial and lateral resolution has the advantage of minimizing unwanted photodamage while still monitoring macroscopic changes to cerebral blood flow during photothrombosis. We show that temporally guiding illumination using real-time feedback of blood flow dynamics also minimized unwanted photodamage to the vascular network. Finally, through quantifiable behavior deficits and chronic imaging we show that this model can be used to study recovery mechanisms or the effects of therapeutics longitudinally.R01 EB021018 - NIBIB NIH HHS; R01 MH111359 - NIMH NIH HHS; R01 NS108472 - NINDS NIH HHSPublished versio

Penta-Modal Imaging Platform with OCT- Guided Dynamic Focusing for Simultaneous Multimodal Imaging

Complex diseases, such as Alzheimer’s disease, are associated with sequences of changes in multiple disease-specific biomarkers. These biomarkers may show dynamic changes at specific stages of disease progression. Thus, testing/monitoring each biomarker may provide insight into specific disease-related processes, which can result in early diagnosis or even development of preventive measures. Obtaining a comprehensive information of biological tissues requires imaging of multiple optical contrasts, which is not typically offered by a single imaging modality. Thus, combining different contrast mechanisms to achieve simultaneous multimodal imaging is desirable. However, this process is highly challenging due to specific optical and hardware requirements for each optical imaging system. The objective of this dissertation is to develop a novel Penta-modal optical imaging system integrating photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT), OCT angiography (OCTA) and confocal fluorescence microscopy (CFM) in one platform providing comprehensive structural, functional, and molecular information of living biological tissues. The system can simultaneously image different biomarkers with a large field-of-view (FOV) and high-speed imaging. The large FOV and the high imaging speed is achieved by combining optical and mechanical scanning mechanisms. To compensate for an uneven surface of biological samples, which result in images with non-uniform resolution and low signal to noise ratio (SNR), we further develop a novel OCT-guided surface contour scanning methodology, a technique for adjusting objective lens focus to follow the contour of the sample surface, to provide a uniform spatial resolution and SNR across the region of interest (ROI). The imaging system was tested by imaging phantoms, ex vivo biological samples, and in vivo. The OCT-guided surface contour scanning methodology was utilized for imaging a leaf of purple queen plant, which resulted in a significant contrast improvement of 41% and 38% across a large imaging area for CFM and PAM, respectively. The nuclei and cells walls were also clearly observed in both images. In an in vivo imaging of the Swiss Webster mouse ear, our multimodal imaging system was able to provide images with uniform resolution in an FOV of 10 mm x 10 mm with an imaging time of around 5 minutes. In addition to measuring the blood flow in the mouse ear, the system also successfully imaged mouse ear blood vessels, sebaceous glands, as well as several tissue structures. We further conducted a comparative study of OCTA for rodent retinal imaging by evaluating the performance of three OCTA algorithms, namely the phase variance (PV), improved speckle contrast (ISC), and optical microangiography (OMAG). It was concluded that the OMAG algorithm provided statistically significant higher mean values of BVD and VPI compared to the ISC algorithm (0.27±0.07 vs. 0.24±0.05 for BVD; 0.09±0.04 and 0.08±0.04 for VPI), while no statistically significant difference was observed for VDI and VCI among the algorithms. Results showed that both the ISC and OMAG algorithms are more robust than PV, and they can reveal similar vasculature features. Lastly, we utilized the proposed imaging system to monitor, for the first time, the invasion process of malaria parasites in the mosquito midgut. The system shows a promising potential to detect parasite motion as well as structural changes inside the mosquito midgut. The multimodal imaging system outlined in this dissertation can be useful in a variety of applications thanks to the specific optical contrast offered by each employed modality, including retinal and brain imaging

Quantitative optical imaging platform for studying neurovascular hemodynamics during ischemic stroke

The measurement of cerebral hemodynamics is vital for the study of physiological and pathophysiological conditions in the brain. Preclinical research examining the mechanisms, outcomes, and efficacies of interventions during ischemic stroke require quantitative imaging technologies. This dissertation presents the development of an optical imaging platform capable of chronic in vivo monitoring of cortical hemodynamics during ischemic stroke and the subsequent recovery. The system combines two different imaging techniques, laser speckle contrast imaging (LSCI) and oxygen-dependent quenching of phosphorescence, to measure cerebral blood flow (CBF) and dissolved oxygen tension (pO2) within cortical vasculature. Phosphorescence signal localization is achieved through the use of a spatial light modulator to selectively pattern excitation light in order to overcome the traditional limitations of the imaging modality. The spatial light modulator is also utilized to perform artery-targeted photothrombosis for the induction of highly-localized, reproducible infarcts as an extension of the photothrombotic model of ischemic stroke. The LSCI hardware implements the multi-exposure speckle imaging (MESI) technique for robust estimates of CBF that facilitate day-to-day and between-subject comparisons. The integration of an awake imaging system eliminates the confounding effects of anesthesia upon cerebral hemodynamics. The capabilities of the complete optical imaging platform are demonstrated by monitoring the acute hemodynamic response during targeted photothrombosis and the chronic recovery of the resulting ischemic infarctBiomedical Engineerin

Modern laser speckle contrast theory: flaws and consequences

Laser speckle contrast imaging (LSCI) is a non-invasive optical imaging technique for monitoring blood flow in brain, skin, and retina. The simple and cheap instrument makes it a promising technology for both clinical applications and research. Modern LSCI theory takes advantage of the relation between blood flow and the speckle contrast v ~ 1/K^2 to provide an online acquisition of a full-field blood flow image. However, the assumptions about the form of field correlation function, static scattering effect, and the coherence factor make interpretation of the contrast imprecise. Here we examined how the assumptions in modern LSCI theory affect the relative blood flow measurement and utilized Dynamic Laser Speckle Imaging (DLSI) to validate the imprecision of modern LSCI. Most importantly, the contrast models for measuring relative flow in the brain parenchyma and the large vessels were derived. It turns out that modern LSCI underestimates blood flow change and leads to significant error for slow blood flow measurement.2020-06-03T00:00:00

In Vivo Vascular Imaging with Photoacoustic Microscopy

Photoacoustic (PA) tomography (PAT) has received extensive attention in the last decade for its capability to provide label-free structural and functional imaging in biological tissue with highly scalable spatial resolution and penetration depth. Compared to modern optical modalities, PAT offers speckle-free images and is more sensitive to optical absorption contrast (with 100% relative sensitivity). By implementing different regimes of optical wavelength, PAT can be used to image diverse light-absorbing biomolecules. For example, hemoglobin is of particular interest in the visible wavelength regime owing to its dominant absorption, and lipids and water are more commonly studied in the near-infrared regime. In this dissertation, one challenge was to quantitatively investigate red-blood-cell dynamics in nailfold capillaries with single-cell resolution PA microscopy (PAM). We recruited healthy volunteers and measured multiple hemodynamic parameters based on individual red blood cells (RBCs). Statistical analysis revealed the process of oxygen release and changes in flow speed for RBCs in a capillary. For the first time on record, oxygen release from individual RBCs in human capillaries was imaged with nearly real-time speed, and the work paved the way for our following study of a specific blood disorder. We next conducted a pilot study on sickle cell disease (SCD), measuring and comparing the parameters related to RBC dynamics between healthy subjects and patients with SCD. In the patient group, we found that capillaries tended to be more tortuous, dilated, and had higher number density. In addition, abnormal RBCs tended to have lower oxygenation in the inlet of a capillary, from where they flowed slower and released a larger fraction of oxygen than normal RBCs. As the only imaging modality able to observe the real-time dynamics of the oxygen release of individual RBCs, PAM provides medically valuable information for diagnostic purposes. As the last focus of this dissertation, we tackled the limited view problem in PAM by introducing an off-axis illumination technique for complementing the original detection view. We demonstrated this technique numerically and then experimentally on phantoms and animals. This simple but very effective method revealed abundant vertical vasculature in a mouse brain that had long been missed by conventional top-illumination PAM. This technique greatly advances future studies on neurovascular responses in mouse brains

Optical Methods in Sensing and Imaging for Medical and Biological Applications

The recent advances in optical sources and detectors have opened up new opportunities for sensing and imaging techniques which can be successfully used in biomedical and healthcare applications. This book, entitled ‘Optical Methods in Sensing and Imaging for Medical and Biological Applications’, focuses on various aspects of the research and development related to these areas. The book will be a valuable source of information presenting the recent advances in optical methods and novel techniques, as well as their applications in the fields of biomedicine and healthcare, to anyone interested in this subject

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

Functional Connectivity of the Rodent Brain Using Optical Imaging

RÉSUMÉ L'objectif de cette thèse de doctorat est d’appliquer la connectivité fonctionnelle dans une variété de modèles animaux, à l’aide de plusieurs techniques d’imagerie optique. Le cerveau, même au repos, montre une activité métabolique élevée : la corrélation des fluctuations spontanées lentes permet d’identifier des régions cérébrales distantes mais connectées; d’où le terme connectivité fonctionnelle. Les changements dans l’activité spontanée peuvent donner un aperçu des processus neuronaux qui comprennent la majorité de l’activité métabolique du cerveau, et constituent en conséquent une vaste source de changements reliés aux maladies. L’hémodynamique du cerveau peut être modifiée grâce à des affections neurovasculaires et avoir un effet sur l’activité au repos. Cette thèse vise la compréhension des changements de connectivité fonctionnelle induits par des maladies, à l’aide de l’imagerie optique fonctionnelle. Les techniques d’imagerie explorées dans les deux premières contributions de cette thèse sont l’Imagerie Optique Intrinsèque et l’Imagerie par Granularité Laser. Ensemble, elles peuvent estimer les changements de consommation d'oxygène, étroitement liés à l’activité neuronale. Ces techniques possèdent des résolutions temporelles et spatiales adéquates et bien adaptées pour imager la convexité du cortex cérébral. Dans le dernier article, une modalité d’imagerie en profondeur, la Tomographie Photoacoustique a été utilisée chez le rat nouveau-né. La Tomographie par Cohérence Optique et la Tomographie Laminaire Optique font également partie de la gamme des techniques d’imagerie développées et appliquées dans d’autres collaborations. La première partie des résultats mesure les changements de connectivité fonctionnelle dans un modèle d’activité épileptiforme aiguë chez le rongeur. Il y a des augmentations ainsi que des diminutions entre les corrélations homologues, avec une faible dépendance aux crises épileptiques. Ces changements suggèrent un découplage potentiel entre les paramètres hémodynamiques dans les réseaux au repos, en soulignant l’importance d’investiguer les réseaux épileptiques à l’aide de plusieurs mesures hémodynamiques indépendantes. La deuxième partie des travaux étudie un nouveau modèle de rigidité artérielle chez la souris : la calcification unilatérale de la carotide droite. L’analyse de connectivité basé sur les régions d’intérêt montre une tendance décroissante de corrélation homologue dans les cortex moteur et cingulum. L’analyse de graphes montre une randomisation des réseaux corticaux, ce qui suggère une perte de connectivité; plus spécifiquement, dans le cortex moteur ipsilateral à la carotide----------ABSTRACT The aim of this thesis is to apply functional connectivity in a variety of animal models, using several optical imaging modalities. Even at rest, the brain shows high metabolic activity: the correlation in slow spontaneous fluctuations identifies remotely connected areas of the brain; hence the term “functional connectivity”. Ongoing changes in spontaneous activity may provide insight into the neural processing that takes most of the brain metabolic activity, and so may provide a vast source of disease related changes. Brain hemodynamics may be modified during disease and affect resting-state activity. The thesis aims to better understand these changes in functional connectivity due to disease, using functional optical imaging. The optical imaging techniques explored in the first two contributions of this thesis are Optical Imaging of Intrinsic Signals and Laser Speckle Contrast Imaging, together they can estimate the metabolic rate of oxygen consumption, that closely parallels neural activity. They both have adequate spatial and temporal resolution and are well adapted to image the convexity of the mouse cortex. In the last article, a depth-sensitive modality called photoacoustic tomography was used in the newborn rat. Optical coherence tomography and laminar optical tomography were also part of the array of imaging techniques developed and applied in other collaborations. The first article of this work shows the changes in functional connectivity in an acute murine model of epileptiform activity. Homologous correlations are both increased and decreased with a small dependence on seizure duration. These changes suggest a potential decoupling between the hemodynamic parameters in resting-state networks, underlining the importance to investigate epileptic networks with several independent hemodynamic measures. The second study examines a novel murine model of arterial stiffness: the unilateral calcification of the right carotid. Seed-based connectivity analysis showed a decreasing trend of homologous correlation in the motor and cingulate cortices. Graph analyses showed a randomization of the cortex functional networks, suggesting a loss of connectivity, more specifically in the motor cortex ipsilateral to the treated carotid; however these changes are not reflected in differentiated metabolic estimates. Confounds remain due to the fact that carotid rigidification gives rise to neural decline in the hippocampus as well as unilateral alteration of vascular pulsatility; howeve

Digging Deeper with Diffuse Correlation Spectroscopy

Patients with neurological diseases are vulnerable to cerebral ischemia, which can lead to brain injury. In the intensive care unit (ICU), neuromonitoring techniques that can detect flow reductions would enable timely administration of therapies aimed at restoring adequate cerebral perfusion, thereby avoiding damage to the brain. However, suitable bedside neuromonitoring methods sensitive to changes of blood flow and/or oxygen metabolism have yet to be established. Near-infrared spectroscopy (NIRS) is a promising technique capable of non-invasively monitoring flow and oxygenation. Specifically, diffuse correlation spectroscopy (DCS) and time-resolved (TR) NIRS can be used to monitor blood flow and tissue oxygenation, respectively, and combined to measuring oxidative metabolism. The work presented in this thesis focused on advancing a DCS/TR-NIRS hybrid system for acquiring these physiological measurements at the bedside. The application of NIRS for neuromonitoring is favourable in the neonatal ICU since the relatively thin scalp and skull of infants has minimal effect on the detected optical signal. Considering this application, the validation of a combined DCS/NIRS method for measuring the cerebral metabolic rate of oxygen (CMRO2) was investigated in Chapter 2. Although perfusion changes measured by DCS have been confirmed by various flow modalities, characterization of photon scattering in the brain is not clearly understood. Chapter 3 presents the first DCS study conducted directly on exposed cortex to confirm that the Brownian motion model is the best flow model for characterizing the DCS signal. Furthermore, a primary limitation of DCS is signal contamination from extracerebral tissues in the adult head, causing CBF to be underestimated. In Chapter 4, a multi-layered model was implemented to separate signal contributions from scalp and brain; derived CBF changes were compared to computed tomography perfusion. Overall, this thesis advances DCS techniques by (i) quantifying cerebral oxygen metabolism, (ii) confirming the more appropriate flow model for analyzing DCS data and (iii) demonstrating the ability of DCS to measure CBF accurately despite the presence of a thick (1-cm) extracerebral layer. Ultimately, the work completed in this thesis should help with the development of a hybrid DCS/NIRS system suitable for monitoring cerebral hemodynamics and energy metabolism in critical-ill patients

Laser speckle based techniques for blood flow estimation in small animal and human brain

Cerebral blood flow (CBF) is a biomarker for brain health, facilitating the advancement of studies on brain states in both healthy and diseased individuals. While there are indirect approaches of CBF based on human physiology, there is a need for technology that measures CBF directly and continuously. Laser speckle contrast imaging (LSCI) is an optical modality that measures changes in CBF by analyzing the blurring of speckle patterns. LSCI has been extensively employed to obtain two-dimensional blood flow maps in thinned-skull mouse brains and has found diverse applications in studies involving the retina, skin, and strokes. However, the effectiveness of LSCI has been limited in animal models due to the lack of depth-sensitivity. Speckle contrast optical spectroscopy (SCOS), an extension of LSCI for non-invasive human brain studies, has recently been developed to probe dynamics in deeper tissue regions by increasing the source-detector separation. But the low photon flux detected from human brain limits the usability of SCOS for brain activation measurements. To address these limitations, this thesis focuses on advancements made in laser speckle technology for improved measure of blood flow in both animal and human brains. Firstly, analytical and numerical methods have been developed for an interferometric LSCI system, which employs a heterodyne detection scheme to enhance CBF within the coherence volume in small animals. Next, a dynamic speckle model (DSM) is created to simulate the temporal evolution of the speckle patterns. DSM has been utilized to quantify the impact of noise sources on speckle contrast, particularly relevant in human brain measurements utilizing SCOS where low photon counts is a norm. Finally, a fiber-based SCOS system with a long source-detector separation has been presented to perform human brain activation studies. Through experiments involving three healthy subjects performing a mental subtraction task, changes in brain activation have been observed. Importantly, the SCOS system has demonstrated an order of magnitude improvement in the signal-to-noise ratio compared to the state-of-the-art diffuse correlation spectroscopy system.These methods serve as valuable tools to augment existing LSCI systems and promoting the widespread adoption of SCOS in human brain activation studies thus contributing to the development of future non-invasive, continuous, and cost-effective blood flow monitoring devices