Faculty Bibliography

In vivo optical imaging of cerebral blood flow (CBF) and metabolism did not exist 50 years ago. While point optical fluorescence and absorption measurements of cellular metabolism and hemoglobin concentrations had already been introduced by then, point blood flow measurements appeared only 40 years ago. The advent of digital cameras has significantly advanced two-dimensional optical imaging of neuronal, metabolic, vascular, and hemodynamic signals. More recently, advanced laser sources have enabled a variety of novel three-dimensional high-spatial-resolution imaging approaches. Combined, as we discuss here, these methods are permitting a multifaceted investigation of the local regulation of CBF and metabolism with unprecedented spatial and temporal resolution. Through multimodal combination of these optical techniques with genetic methods of encoding optical reporter and actuator proteins, the future is bright for solving the mysteries of neurometabolic and neurovascular coupling and translating them to clinical utility.

In vivo optical microscopic imaging techniques have recently emerged as important tools for the study of neurobiological development and pathophysiology. In particular, two-photon microscopy has proved to be a robust and highly flexible method for in vivo imaging in highly scattering tissue. However, two-photon imaging typically requires extrinsic dyes or contrast agents, and imaging depths are limited to a few hundred microns. Here we demonstrate Optical Coherence Microscopy (OCM) for in vivo imaging of neuronal cell bodies and cortical myelination up to depths of ~1.3 mm in the rat neocortex. Imaging does not require the administration of exogenous dyes or contrast agents, and is achieved through intrinsic scattering contrast and image processing alone. Furthermore, using OCM we demonstrate in vivo, quantitative measurements of optical properties (index of refraction and attenuation coefficient) in the cortex, and correlate these properties with laminar cellular architecture determined from the images. Lastly, we show that OCM enables direct visualization of cellular changes during cell depolarization and may therefore provide novel optical markers of cell viability.

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.

Organs such as the heart and brain possess intricate fiber structures that are best characterized with three-dimensional imaging. For instance, diffusion-based, magnetic resonance tractography (MRT) enables studies of connectivity and remodeling during development and disease macroscopically on the millimeter scale. Here we present complementary, high-resolution microscopic optical coherence imaging and analysis methods that, when used in conjunction with clearing techniques, can characterize fiber architecture in intact organs at tissue depths exceeding 1 mm. We anticipate that these techniques can be used to study fiber architecture in situ at microscopic scales not currently accessible to diffusion magentic resonance (MR), and thus, to validate and complement macroscopic structural imaging techniques. Moreover, as these techniques use intrinsic signals and do not require tissue slicing and staining, they can be used for high-throughput, nondestructive evaluation of fiber architecture across large tissue volumes.

Doppler optical coherence tomography (DOCT) and OCT angiography are novel methods to investigate cerebrovascular physiology. In the rodent cortex, DOCT flow displays features characteristic of cerebral blood flow, including conservation along nonbranching vascular segments and at branch points. Moreover, DOCT flow values correlate with hydrogen clearance flow values when both are measured simultaneously. These data validate DOCT as a noninvasive quantitative method to measure tissue perfusion over a physiologic range.

PURPOSE/OBJECTIVE:The purpose of this study was to report on a posterior segment coloboma manifesting unusual morphology as determined by high-speed, ultrahigh-resolution optical coherence tomography imaging. METHODS:A 47-year-old woman with bilateral colobomas was evaluated by fundus examination and high-speed, ultrahigh-resolution optical coherence tomography imaging. RESULTS:Imaging with high-speed, ultrahigh-resolution optical coherence tomography showed intact retinal pigment epithelium within the posterior segment coloboma. Most of the retinal layers seemed to continue into the coloboma, although they exhibited slight attenuation. The external limiting membrane was clearly visible continuing within the coloboma, suggesting that Müller cells and the inner segments of the photoreceptors were still present in this area. The junction between the inner and outer segments of the photoreceptors ended at the margin of the coloboma, which may be because of either photoreceptor disruption or a change in the orientation of the outer segments. CONCLUSION/CONCLUSIONS:High-speed, ultrahigh-resolution optical coherence tomography imaging showed the presence of Müller cells and photoreceptor inner segments within a posterior segment coloboma. The retinal pigment epithelium was intact within the coloboma, representing an unusual morphology.

Measuring cerebral oxygen delivery and metabolism microscopically is important for interpreting macroscopic functional magnetic resonance imaging (fMRI) data and identifying pathological changes associated with stroke, Alzheimer's disease, and brain injury. Here, we present simultaneous, microscopic measurements of cerebral blood flow (CBF) and oxygen partial pressure (pO(2)) in cortical microvessels of anesthetized rats under baseline conditions and during somatosensory stimulation. Using a custom-built imaging system, we measured CBF with Fourier-domain optical coherence tomography (OCT), and vascular pO(2) with confocal phosphorescence lifetime microscopy. Cerebral blood flow and pO(2) measurements displayed heterogeneity over distances irresolvable with fMRI and positron emission tomography. Baseline measurements indicate O(2) extraction from pial arterioles and homogeneity of ascending venule pO(2) despite large variation in microvessel flows. Oxygen extraction is linearly related to flow in ascending venules, suggesting that flow in ascending venules closely matches oxygen demand of the drained territory. Oxygen partial pressure and relative CBF transients during somatosensory stimulation further indicate arteriolar O(2) extraction and suggest that arterioles contribute to the fMRI blood oxygen level dependent response. Understanding O(2) supply on a microscopic level will yield better insight into brain function and the underlying mechanisms of various neuropathologies.

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury. Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.

We suggest that Diffuse Correlation Spectroscopy (DCS) measurements of tissue blood flow primarily probe relative red blood cell (RBC) motion, due to the occurrence of multiple sequential scattering events within blood vessels. The magnitude of RBC shear-induced diffusion is known to correlate with flow velocity, explaining previous reports of linear scaling of the DCS "blood flow index" with tissue perfusion despite the observed diffusion-like auto-correlation decay. Further, by modeling RBC mean square displacement using a formulation that captures the transition from ballistic to diffusive motion, we improve the fit to experimental data and recover effective diffusion coefficients and velocity de-correlation time scales in the range expected from previous blood rheology studies.