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.

Measurements of oxygen partial pressure (pO(2)) with high temporal and spatial resolution in three dimensions is crucial for understanding oxygen delivery and consumption in normal and diseased brain. Among existing pO(2) measurement methods, phosphorescence quenching is optimally suited for the task. However, previous attempts to couple phosphorescence with two-photon laser scanning microscopy have faced substantial difficulties because of extremely low two-photon absorption cross-sections of conventional phosphorescent probes. Here we report to our knowledge the first practical in vivo two-photon high-resolution pO(2) measurements in small rodents' cortical microvasculature and tissue, made possible by combining an optimized imaging system with a two-photon-enhanced phosphorescent nanoprobe. The method features a measurement depth of up to 250 microm, sub-second temporal resolution and requires low probe concentration. The properties of the probe allowed for direct high-resolution measurement of cortical extravascular (tissue) pO(2), opening many possibilities for functional metabolic brain studies.

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.

What is the organization of cerebral microvascular oxygenation and morphology that allows adequate tissue oxygenation at different activity levels? We address this question in the mouse cerebral cortex using microscopic imaging of intravascular O2 partial pressure and blood flow combined with numerical modelling. Here we show that parenchymal arterioles are responsible for 50% of the extracted O2 at baseline activity, and the majority of the remaining O2 exchange takes place within the first few capillary branches. Most capillaries release little O2 at baseline acting as an O2 reserve that is recruited during increased neuronal activity or decreased blood flow. Our results challenge the common perception that capillaries are the major site of O2 delivery to cerebral tissue. The understanding of oxygenation distribution along arterio-capillary paths may have profound implications for the interpretation of blood-oxygen-level dependent (BOLD) contrast in functional magnetic resonance imaging and for evaluating microvascular O2 delivery capacity to support cerebral tissue in disease.

The blood oxygenation level-dependent (BOLD) contrast is widely used in functional magnetic resonance imaging (fMRI) studies aimed at investigating neuronal activity. However, the BOLD signal reflects changes in blood volume and oxygenation rather than neuronal activity per se. Therefore, understanding the transformation of microscopic vascular behavior into macroscopic BOLD signals is at the foundation of physiologically informed noninvasive neuroimaging. Here, we use oxygen-sensitive two-photon microscopy to measure the BOLD-relevant microvascular physiology occurring within a typical rodent fMRI voxel and predict the BOLD signal from first principles using those measurements. The predictive power of the approach is illustrated by quantifying variations in the BOLD signal induced by the morphological folding of the human cortex. This framework is then used to quantify the contribution of individual vascular compartments and other factors to the BOLD signal for different magnet strengths and pulse sequences.