Pia-FLOW: Deciphering hemodynamic maps of the pial vascular connectome and its response to arterial occlusion.

The pial vasculature is the sole source of blood supply to the neocortex. The brain is contained within the skull, a vascularized bone marrow with a unique anatomical connection to the brain meninges. Recent developments in tissue clearing have enabled detailed mapping of the entire pial and calvarial vasculature. However, what are the absolute flow rate values of those vascular networks? This information cannot accurately be retrieved with the commonly used bioimaging methods. Here, we introduce Pia-FLOW, a unique approach based on large-scale transcranial fluorescence localization microscopy, to attain hemodynamic imaging of the whole murine pial and calvarial vasculature at frame rates up to 1,000 Hz and spatial resolution reaching 5.4 µm. Using Pia-FLOW, we provide detailed maps of flow velocity, direction, and vascular diameters which can serve as ground-truth data for further studies, advancing our understanding of brain fluid dynamics. Furthermore, Pia-FLOW revealed that the pial vascular network functions as one unit for robust allocation of blood after stroke.

Cortex-wide transcranial localization microscopy with fluorescently labeled red blood cells.

Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease.

Depth-Resolved Localization Microangiography in the NIR-II Window.

Detailed characterization of microvascular alterations requires high-resolution 3D imaging methods capable of providing both morphological and functional information. Existing optical microscopy tools are routinely used for microangiography, yet offer suboptimal trade-offs between the achievable field of view and spatial resolution with the intense light scattering in biological tissues further limiting the achievable penetration depth. Herein, a new approach for volumetric deep-tissue microangiography based on stereovision combined with super-resolution localization imaging is introduced that overcomes the spatial resolution limits imposed by light diffusion and optical diffraction in wide-field imaging configurations. The method capitalizes on localization and tracking of flowing fluorescent particles in the second near-infrared window (NIR-II, ≈1000-1700 nm), with the third (depth) dimension added by triangulation and stereo-matching of images acquired with two short-wave infrared cameras operating in a dual-view mode. The 3D imaging capability enabled with the proposed method facilitates a detailed visualization of microvascular networks and an accurate blood flow quantification. Experiments performed in tissue-mimicking phantoms demonstrate that high resolution is preserved up to a depth of 4 mm in a turbid medium. Transcranial microangiography of the entire murine cortex and penetrating vessels is further demonstrated at capillary level resolution.

Digital aberration correction enhances field of view in visible-light optical coherence microscopy.

In optical coherence microscopy, optical aberrations commonly result in astigmatism-dominated wavefront errors in the peripheral regions of the optical objective, primarily elongating the microscope's point-spread function along the radial direction in the vicinity of the focal plane. We report on enhanced-field-of-view optical coherence microscopy through computational aberration correction in the visible-light range. An isotropic spatial resolution of 2.5 µm was achieved over an enhanced lateral field of view spanning 1.3 mm × 1.6 mm, as experimentally verified in a micro-bead phantom and further demonstrated in ex vivo tissue samples. The extended field of view achieved by the digital aberration correction facilitates the use of low-cost systems by averting the need for high-quality objectives.

Concept for Markerless 6D Tracking Employing Volumetric Optical Coherence Tomography.

Optical tracking systems are widely used, for example, to navigate medical interventions. Typically, they require the presence of known geometrical structures, the placement of artificial markers, or a prominent texture on the target's surface. In this work, we propose a 6D tracking approach employing volumetric optical coherence tomography (OCT) images. OCT has a micrometer-scale resolution and employs near-infrared light to penetrate few millimeters into, for example, tissue. Thereby, it provides sub-surface information which we use to track arbitrary targets, even with poorly structured surfaces, without requiring markers. Our proposed system can shift the OCT's field-of-view in space and uses an adaptive correlation filter to estimate the motion at multiple locations on the target. This allows one to estimate the target's position and orientation. We show that our approach is able to track translational motion with root-mean-squared errors below 0 . 25 m m and in-plane rotations with errors below 0 . 3 ∘ . For out-of-plane rotations, our prototypical system can achieve errors around 0 . 6 ∘ .