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.