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.
Keywords: fluorescence microscopy; localization imaging; microangiography; second near-infrared spectrum; stereovision.
© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.