In this work, we demonstrate the application of Rose Bengal as a photo-thrombotic agent using 2-photon and 3-photon excitation by measuring the response to multiphotonic excitation. We also demonstrate, in the case of 2-photon excitation, that the mechanism used is different from laser injury alone with a control group of FitC injected mice. Preliminary results show that a capillary photo-thrombosis could be performed up to 200 μm and OCT imaging could confirm blockage.
As red blood cells flowing in capillaries pass through the focal volume of an optical coherence tomography (OCT) system, they will induce a transient change in back-scattering, leading to peaks in the intensity of the OCT signal. In recent years, an OCT method has been devised exploiting these intensity fluctuations to estimate the hemodynamic parameters, such as RBC speed and flux. In this work, using concurrent OCT and two-photon microscopy, we assess the accuracy of the technique, highlighting that single red blood cells can indeed be detected in OCT, in specific hemodynamic and experimental settings.
Significance: Understanding how the brain recovers from cerebral tissue and vascular damage after an ischemic event can help develop new therapeutic strategies for the treatment of stroke.
Aim: We investigated cerebral tissue repair and microvasculature regeneration and function after a targeted ischemic stroke.
Approach: Following photothrombosis occlusion of microvasculature, chronic optical coherence tomography (OCT)-based angiography was used to track ischemic tissue repair and microvasculature regeneration at three different cortical depths and up to 28 days in awake animals. Capillary network orientation analysis was performed to study the structural pattern of newly formed microvasculature. Based on the time-resolved OCT-angiography, we also investigated capillary stalling, which is likely related to ischemic stroke-induced inflammation.
Results: Deeper cerebral tissue was found to have a larger ischemic area than shallower regions at any time point during the course of poststroke recovery, which suggests that cerebral tissue located deep in the cortex is more vulnerable. Regenerated microvasculature had a highly organized pattern at all cortical depths with a higher degree of structural reorganization in deeper regions. Additionally, capillary stalling event analysis revealed that cerebral ischemia augmented stalling events considerably.
Conclusion: Longitudinal OCT angiography reveals that regenerated capillary network has a highly directional pattern and an increased density and incidence of capillary stalling event.
KEYWORDS: Two photon excitation microscopy, In vivo imaging, Blood, 3D acquisition, 3D image processing, Optical coherence tomography, Hemodynamics, Interferometry, Blood circulation, Angiography
Over the past decades, several techniques have been developed to measure cerebral hemodynamic parameters such as red blood cell velocity, flux and hematocrit using optical coherence tomography (OCT). Despite these advancements, two photon microscopy (TPM) remains the gold standard for functional cerebral hemodynamic imaging, though localized line-scan or bolus tracking. Here we developed an optical platform enabling concurrent imaging with OCT and TPM and perform co-localized and simultaneous velocimetric measurements with both modalities, with the aim of validating OCT velocimetric methods.
Over the past decades, optical coherence tomography has emerged as an important imaging technique to study biological processes through its ability to perform three-dimensional imaging at high acquisition rates and non-invasively. Furthermore, OCT has shown a growing interest in brain imaging through its capacity in obtaining functional information such as cellular viability, hematocrit and blood flow velocity.
Although OCT can reach image depths spanning a few millimeters, the effective imaging depth is typically dictated by the depth-of-field of the imaging optics. In traditional OCT systems, this depth-of-field is given by the Rayleigh range and is thus coupled to the lateral resolution. As such, increasing the numerical aperture of the system reduces the imaging depth, ultimately hampering the depth-multiplexing advantage of OCT. Wavefront engineering schemes have been devised to overcome this limitation, providing the OCT systems with an extended-focus. We present here two extended-focus OCT systems (xf-OCT) optimized for cerebral imaging. The first system operates in the visible wavelength range and is designed to image the superficial cortex of mice at high contrast and at high resolution. Its high axial and lateral resolution of 0.8 and 1.4 um respectively, maintained over 200 um enable resolving structures such as myelinated axons, neuronal cells and micro-vessels in vivo. The second system is optimized for deep microvascular cortical imaging and operates in the infrared spectral range. Through its extended-focus and increased penetration, the second system can provide maps of cortical microvasculature over 800 um in depth in the cortex in vivo.
Visible light optical coherence tomography has shown great interest in recent years for spectroscopic and high-resolution retinal and cerebral imaging. Here, we present an extended-focus optical coherence microscopy system operating from the visible to the near-infrared wavelength range for high axial and lateral resolution imaging of cortical structures in vivo. The system exploits an ultrabroad illumination spectrum centered in the visible wavelength range (λc = 650 nm, Δλ ∼ 250 nm) offering a submicron axial resolution (∼0.85 μm in water) and an extended-focus configuration providing a high lateral resolution of ∼1.4 μm maintained over ∼150 μm in depth in water. The system’s axial and lateral resolution are first characterized using phantoms, and its imaging performance is then demonstrated by imaging the vasculature, myelinated axons, and neuronal cells in the first layers of the somatosensory cortex of mice in vivo.
Magnetic Resonance Imaging has revolutionised our understanding of brain function through its ability to image human cerebral structures non-invasively over the entire brain. By exploiting the different magnetic properties of oxygenated and deoxygenated blood, functional MRI can indirectly map areas undergoing neural activation. Alongside the development of fMRI, powerful statistical tools have been developed in an effort to shed light on the neural pathways involved in processing of sensory and cognitive information. In spite of the major improvements made in fMRI technology, the obtained spatial resolution of hundreds of microns prevents MRI in resolving and monitoring processes occurring at the cellular level. In this regard, Optical Coherence Microscopy is an ideal instrumentation as it can image at high spatio-temporal resolution. Moreover, by measuring the mean and the width of the Doppler spectra of light scattered by moving particles, OCM allows extracting the axial and lateral velocity components of red blood cells. The ability to assess quantitatively total blood velocity, as opposed to classical axial velocity Doppler OCM, is of paramount importance in brain imaging as a large proportion of cortical vascular is oriented perpendicularly to the optical axis. We combine here quantitative blood flow imaging with extended-focus Optical Coherence Microscopy and Statistical Parametric Mapping tools to generate maps of stimuli-evoked cortical hemodynamics at the capillary level.
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