Time-resolved laser speckle contrast imaging (TR-LSCI) of cerebral blood flow

To address many of the deficiencies in optical neuroimaging technologies such as poor spatial resolution, time-consuming reconstruction, low penetration depth, and contact-based measurement, a novel, noncontact, time-resolved laser speckle contrast imaging (TR-LSCI) technique has been developed for continuous, fast, and high-resolution 2D mapping of cerebral blood flow (CBF) at different depths of the head. TR-LSCI illuminates the head with picosecond-pulsed, coherent, widefield near-infrared light and synchronizes a newly developed, high-resolution, gated single-photon avalanche diode camera (SwissSPAD2) to capture CBF maps at different depths. By selectively collecting diffuse photons with longer pathlengths through the head, TR-LSCI reduces partial volume artifacts from the overlying tissues, thus improving the accuracy of CBF measurement in the deep brain. CBF map reconstruction was dramatically expedited by incorporating highly parallelized computation. The performance of TR-LSCI was evaluated using head-simulating phantoms with known properties and in-vivo rodents with varied hemodynamic challenges to the brain. Results from these pilot studies demonstrated that TR-LSCI enabled mapping CBF variations at different depths with a sampling rate of up to 1 Hz and spatial resolutions ranging from tens of micrometers on the head surface to 1-2 millimeters in the deep brain. With additional improvements and validation in larger populations against established methods, we anticipate offering a noncontact, fast, high-resolution, portable, and affordable brain imager for fundamental neuroscience research in animals and for translational studies in humans.Comment: 22 pages, 7 figures, 4 table

High-density speckle contrast optical tomography of cerebral blood flow response to functional stimuli in the rodent brain

Noninvasive, three-dimensional, and longitudinal imaging of cerebral blood flow (CBF) in small animal models and ultimately in humans has implications for fundamental research and clinical applications. It enables the study of phenomena such as brain development and learning and the effects of pathologies, with a clear vision for translation to humans. Speckle contrast optical tomography (SCOT) is an emerging optical method that aims to achieve this goal by directly measuring three-dimensional blood flow maps in deep tissue with a relatively inexpensive and simple system. High-density SCOT is developed to follow CBF changes in response to somatosensory cortex stimulation. Measurements are carried out through the intact skull on the rat brain. SCOT is able to follow individual trials in each brain hemisphere, where signal averaging resulted in comparable, cortical images to those of functional magnetic resonance images in spatial extent, location, and depth. Sham stimuli are utilized to demonstrate that the observed response is indeed due to local changes in the brain induced by forepaw stimulation. In developing and demonstrating the method, algorithms and analysis methods are developed. The results pave the way for longitudinal, nondestructive imaging in preclinical rodent models that can readily be translated to the human brain

High-density speckle contrast optical tomography of cerebral blood flow response to functional stimuli in the rodent brain

Noninvasive, three-dimensional, and longitudinal imaging of cerebral blood flow (CBF) in small animal models and ultimately in humans has implications for fundamental research and clinical applications. It enables the study of phenomena such as brain development and learning and the effects of pathologies, with a clear vision for translation to humans. Speckle contrast optical tomography (SCOT) is an emerging optical method that aims to achieve this goal by directly measuring three-dimensional blood flow maps in deep tissue with a relatively inexpensive and simple system. High-density SCOT is developed to follow CBF changes in response to somatosensory cortex stimulation. Measurements are carried out through the intact skull on the rat brain. SCOT is able to follow individual trials in each brain hemisphere, where signal averaging resulted in comparable, cortical images to those of functional magnetic resonance images in spatial extent, location, and depth. Sham stimuli are utilized to demonstrate that the observed response is indeed due to local changes in the brain induced by forepaw stimulation. In developing and demonstrating the method, algorithms and analysis methods are developed. The results pave the way for longitudinal, nondestructive imaging in preclinical rodent models that can readily be translated to the human brain.This project was funded by Fundació CELLEX Barcelona, Ministerio de Economía y Competitividad/FEDER (PHOTODEMENTIA, DPI2015-64358-C2-1-R), Instituto de Salud Carlos III/FEDER (MEDPHOTAGE, DTS16/00087), the “Severo Ochoa” Program for Centers of Excellence in R\&D (SEV-2015-0522), the Obra Social “la Caixa” Foundation (LlumMedBcn), AGAUR-Generalitat (2017 SGR 1380), LASERLAB-EUROPE IV, and “Fundació La Marató TV3.

Compact Low-Cost Fiberless Diffuse Speckle Contrast Flow-Oximeter

A low-cost compact fiberless diffuse speckle contrast flow-meter includes a small laser diode and a 2-dimensional imaging device such as a charge coupled device (CCD) or complementary metal oxide semiconductor (CMOS) for directly contacting the tissue and measuring a parameter such as blood flow in a deep/thick volume of tissue (up to 10 mm depth). The small laser diode is fixed at a certain distance (0 to 20 mm) from the imaging device and directly contacts the tissue. Light emitted from the laser diode penetrates through the tissue and reflects back to the imaging device without passing through any lenses or fibers. One or more additional laser diodes may be added for producing light at different wavelengths, and the combination of measurements taken by the imaging device at the different wavelengths allows for measuring additional parameters, such as both blood flow and blood oxygenation

Scanning, non-contact, hybrid broadband diffuse optical spectroscopy and diffuse correlation spectroscopy system

A scanning system for small animal imaging using non-contact, hybrid broadband diffuse optical spectroscopy (ncDOS) and diffuse correlation spectroscopy (ncDCS) is presented. The ncDOS uses a two-dimensional spectrophotometer retrieving broadband (610-900 nm) spectral information from up to fifty-seven source-detector distances between 2 and 5 mm. The ncDCS data is simultaneously acquired from four source-detector pairs. The sample is scanned in two dimensions while tracking variations in height. The system has been validated with liquid phantoms, demonstrated in vivo on a human fingertip during an arm cuff occlusion and on a group of mice with xenoimplanted renal cell carcinoma. (C) 2016 Optical Society of Americ

Interferometric speckle visibility spectroscopy (ISVS) for human cerebral blood flow monitoring

Infrared light scattering methods have been developed and employed to non-invasively monitor human cerebral blood flow (CBF). However, the number of reflected photons that interact with the brain is low when detecting blood flow in deep tissue. To tackle this photon-starved problem, we present and demonstrate the idea of interferometric speckle visibility spectroscopy (ISVS). In ISVS, an interferometric detection scheme is used to boost the weak signal light. The blood flow dynamics are inferred from the speckle statistics of a single frame speckle pattern. We experimentally demonstrated the improvement of measurement fidelity by introducing interferometric detection when the signal photon number is insufficient. We apply the ISVS system to monitor the human CBF in situations where the light intensity is $\sim$100-fold less than that in common diffuse correlation spectroscopy (DCS) implementations. Due to the large number of pixels ($\sim 2\times 10^5$) used to capture light in the ISVS system, we are able to collect a similar number of photons within one exposure time as in normal DCS implementations. Our system operates at a sampling rate of 100 Hz. At the exposure time of 2 ms, the average signal photon electron number is $\sim$0.95 count/pixel, yielding a single pixel interferometric measurement signal-to-noise ratio (SNR) of $\sim$0.97. The total $\sim 2\times 10^5$ pixels provide an expected overall SNR of 436. We successfully demonstrate that the ISVS system is able to monitor the human brain pulsatile blood flow, as well as the blood flow change when a human subject is doing a breath holding task

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

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

Tissue Damage Characterization Using Non-invasive Optical Modalities

The ability to determine the degree of cutaneous and subcutaneous tissue damage is essential for proper wound assessment and a significant factor for determining patient treatment and morbidity. Accurate characterization of tissue damage is critical for a number of medical applications including surgical removal of nonviable tissue, severity assessment of subcutaneous ulcers, and depth assessment of visually open wounds. The main objective of this research was to develop a non-invasive method for identifying the extent of tissue damage underneath intact skin that is not apparent upon visual examination. This work investigated the relationship between tissue optical properties, blood flow, and tissue viability by testing the hypotheses that (a) changes in tissue oxygenation and/or microcirculatory blood flow measurable by Diffuse Near Infrared Spectroscopy (DNIRS) and Diffuse Correlation Spectroscopy (DCS) differ between healthy and damaged tissue and (b) the magnitude of those changes differs for different degrees of tissue damage. This was accomplished by developing and validating a procedure for measuring microcirculatory blood flow and tissue oxygenation dynamics at multiple depths (up to 1 centimeter) using non-invasive DCS and DNIRS technologies. Due to the lack of pressure ulcer animal models that are compatible with our optical systems, a proof of concept was conducted in a porcine burn model prior to conducting clinical trials in order to assess the efficacy of the system in-vivo. A reduction in total hemoglobin was observed for superficial (5%) and deep burns (35%) along with a statistically significant difference between the optical properties of superficial and deep burns (p < 0.05). Burn depth and viable vessel density were estimated via histological samples. 42% of vessels in the dermal layer were viable for superficial burns, compared to 25% for deep burns. The differences detected in optical properties and hemoglobin content by optical measurements correlated with the extent of tissue injury observed in histological stains. After proof of concept in animals, a human study was conducted and optical data was collected from 20 healthy subjects and 8 patients at risk of developing pressure ulcers. Blood flow index (BFI) values from the sacral region of patients were compared with those of healthy volunteers. Prior to loading measurements, baseline BFI values were measured in subjects in lateral position. These values were systematically higher for patients who developed open ulcers than for the other research subjects. While under the loading position, patients who developed a pressure ulcer had a decrease in BFI from baseline values an order of magnitude larger than healthy subjects (p < 0.01) and patients whose redness dissipated (p < 0.01). The hyperemic response, when pressure was released as the patient was moved back to a lateral position, showed a decreasing trend from one session to the next for patients who developed open ulcers. Overall, this work presents a novel non-invasive method of pressure ulcer assessment and provides an improvement over current assessment methods. The obtained results suggest the system may potentially predict whether non-blanchable redness will develop into an advanced pressure ulcer within four weeks from initial observation.Ph.D., Biomedical Engineering -- Drexel University, 201

On the Indeterminates of Glaucoma:the Controversy of Arterial Blood Pressure and Retinal Perfusion

Glaucoma is a chronic eye disease characterized by thinning of the retina, death of ganglion cells, and progressive loss of vision, eventually leading to blindness. The prevalence of glaucoma is estimated at 1-3% of those over 40 years old. With a constantly aging population, this number is expected to increase significantly over the next 10 years. Even with treatment, about 15% of people with glaucoma currently develop residual vision or tunnel vision and eventually become blind or partially sighted. The mechanisms behind ganglion cell death are poorly understood. Elevated eye pressure is the main risk factor for glaucoma, but treatment in the form of medication, laser, or surgery can only slow the decline, not stop it. In addition, high intraocular pressure is neither necessary nor sufficient for the development of glaucoma, indicating the existence of other unknown risk factors. It has been established that the death of ganglion cells results in a decreased oxygen demand and a concomitant decrease in blood flow. However, there is also a hypothesis that reduced or unstable blood supply is not only a consequence, but also a cause of glaucoma. This is known as the ‘chicken-egg’ dilemma in glaucoma. It is supported by the observation that the risk of developing glaucoma is higher in people with very low blood pressure (sometimes even as a result of overtreatment of high blood pressure).This dissertation is an attempt to methodically examine whether blood pressure can be linked to changes in the retina that could suggest susceptibility to glaucoma. For this purpose, we analyze epidemiological data from the Groningen Longitudinal Glaucoma Study, we use advanced imaging techniques to model the microcirculation, and we describe its relationship with the neural structure and oxygen consumption of the retina. We provide evidence leaning towards the existence of a vascular component, likely pertinent to glaucoma