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Abstract In this study, we imaged the same penetrating cortical vessels in a mouse using ultrahigh field single-vessel MRI at 14 T and high-resolution optical microscopy imaging. The optical imaging was performed through a chronic sealed cranial window, while the single-vessel MRI was facilitated by a custom-designed, chronically implanted radiofrequency coil surrounding the window. The MRI and optical imaging were performed sequentially focused on the same penetrating cortical arterioles and surfacing venules within the whisker barrel cortex. With MRI, we obtained high-resolution multi-gradient echo (MGE) images and single-vessel phase contrast (PC) velocity maps. With optical imaging, we acquired microvascular angiograms using 2-Photon Microscopy (2PM) and Optical Coherence Tomography (OCT) and measured the blood flow velocity using Dynamic Light Scattering OCT (DLS-OCT). The MGE images, PC-based MRI velocity maps, OCT angiograms, and DLS-OCT velocity maps were coregistered with the 2PM microvascular angiograms. Using these tools, we cross-validated blood flow velocity in the penetrating cortical arterioles and surfacing venules measured by single-vessel MRI and OCT at rest. Our novel method demonstrates the possibility of combining ultrahigh field single-vessel MRI and high-resolution optical methods (e.g., 2PM and OCT) for studying brain structure and function with single microvessel precision.