Multiparametric, Longitudinal Optical Coherence Tomography Imaging Reveals Acute Injury and Chronic Recovery in Experimental Ischemic Stroke

Progress in experimental stroke and translational medicine could be accelerated by high-resolution in vivo imaging of disease progression in the mouse cortex. Here, we introduce optical microscopic methods that monitor brain injury progression using intrinsic optical scattering properties of cortical tissue. A multi-parametric Optical Coherence Tomography (OCT) platform for longitudinal imaging of ischemic stroke in mice, through thinned-skull, reinforced cranial window surgical preparations, is described. In the acute stages, the spatiotemporal interplay between hemodynamics and cell viability, a key determinant of pathogenesis, was imaged. In acute stroke, microscopic biomarkers for eventual infarction, including capillary non-perfusion, cerebral blood flow deficiency, altered cellular scattering, and impaired autoregulation of cerebral blood flow, were quantified and correlated with histology. Additionally, longitudinal microscopy revealed remodeling and flow recovery after one week of chronic stroke. Intrinsic scattering properties serve as reporters of acute cellular and vascular injury and recovery in experimental stroke. Multi-parametric OCT represents a robust in vivo imaging platform to comprehensively investigate these properties

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

Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents

Intact in vivo visualization of telencephalic microvasculature in medaka using optical coherence tomography

To date, various human disease models in small fish—such as medaka (Oryzias lapties)--have been developed for medical and pharmacological studies. Although genetic and environmental homogeneities exist, disease progressions can show large individual differences in animal models. In this study, we established an intact in vivo angiographic approach and explored vascular networks in the telencephalon of wild-type adult medaka using the spectral-domain optical coherence tomography. Our approach, which required neither surgical operations nor labeling agents, allowed to visualize blood vessels in medaka telencephala as small as about 8 µm, that is, almost the size of the blood cells of medaka. Besides, we could show the three-dimensional microvascular distribution in the medaka telencephalon. Therefore, the intact in vivo imaging via optical coherence tomography can be used to perform follow-up studies on cerebrovascular alterations in metabolic syndrome and their associations with neurodegenerative disease models in medaka

OCT methods for capillary velocimetry

To date, two main categories of OCT techniques have been described for imaging hemodynamics: Doppler OCT and OCT angiography. Doppler OCT can measure axial velocity profiles and flow in arteries and veins, while OCT angiography can determine vascular morphology, tone, and presence or absence of red blood cell (RBC) perfusion. However, neither method can quantify RBC velocity in capillaries, where RBC flow is typically transverse to the probe beam and single-file. Here, we describe new methods that potentially address these limitations. Firstly, we describe a complex-valued OCT signal in terms of a static scattering component, dynamic scattering component, and noise. Secondly, we propose that the time scale of random fluctuations in the dynamic scattering component are related to red blood cell velocity. Analysis was performed along the slow axis of repeated B-scans to parallelize measurements. We correlate our purported velocity measurements against two-photon microscopy measurements of RBC velocity, and investigate changes during hypercapnia. Finally, we image the ischemic stroke penumbra during distal middle cerebral artery occlusion (dMCAO), where OCT velocimetry methods provide additional insight that is not afforded by either Doppler OCT or OCT angiography

Optical imaging in vivo with a focus on paediatric disease: technical progress, current preclinical and clinical applications and future perspectives

To obtain information on the occurrence and location of molecular events as well as to track target-specific probes such as antibodies or peptides, drugs or even cells non-invasively over time, optical imaging (OI) technologies are increasingly applied. Although OI strongly contributes to the advances made in preclinical research, it is so far, with the exception of optical coherence tomography (OCT), only very sparingly applied in clinical settings. Nevertheless, as OI technologies evolve and improve continuously and represent relatively inexpensive and harmful methods, their implementation as clinical tools for the assessment of children disease is increasing. This review focuses on the current preclinical and clinical applications as well as on the future potential of OI in the clinical routine. Herein, we summarize the development of different fluorescence and bioluminescence imaging techniques for microscopic and macroscopic visualization of microstructures and biological processes. In addition, we discuss advantages and limitations of optical probes with distinct mechanisms of target-detection as well as of different bioluminescent reporter systems. Particular attention has been given to the use of near-infrared (NIR) fluorescent probes enabling observation of molecular events in deeper tissue

Three-dimensional combined photoacoustic and optical coherence microscopy for in vivo microcirculation studies

Photoacoustic microscopy is predominantly sensitive to optical absorption, while optical coherence tomography relies on optical backscattering. Integrating their complementary contrasts can provide comprehensive information about biological tissue. We have developed a dual-modality microscope that combines the two for studying microcirculation. Three-dimensional imaging of microvasculature and its local environment has been demonstrated at micrometer-order resolution using endogenous contrast in vivo

Depth-resolved microscopy of cortical hemodynamics with optical coherence tomography

We describe depth-resolved microscopy of cortical hemodynamics with high-speed spectral/Fourier domain optical coherence tomography (OCT). Stimulus-evoked changes in blood vessel diameter, flow, and total hemoglobin were measured in the rat somatosensory cortex. The results show OCT measurements of hemodynamic changes during functional activation and represent an important step toward understanding functional hyperemia at the microscopic level.National Institutes of Health (U.S.) (R01-NS057476)National Institutes of Health (U.S.) (P01NS055104)National Institutes of Health (U.S.) (P50NS010828)National Institutes of Health (U.S.) (K99NS067050)National Institutes of Health (U.S.) (R01-CA075289-12)United States. Air Force Office of Scientific Research (FA9550-07-1-0014)United States. Dept. of Defense. Medical Free Electron Laser Program (FA9550-07-1-0101