(a) Single coronal KESM section of the mouse cerebellum and mid-brain and closeup showing a (b) 500μm and (c) 200μm view of the same section. (d) A single tissue cross-section at any depth z is composed of several adjacent sections with a width that falls within the objective FOV.

Knife-edge scanning microscopy. Serial sections are concurrently cut and imaged under water (a–b). Imaging is performed in transmission mode by sending illumination through a diamond cutting tool. Image capture is synchronized with stage position and performed using a line-scan camera. Illumination is provided using a mercury vapor short arc lamp to send light to the knife through a liquid-optic light guide (0.55NA, η = 1.65). Geometric angles of the refracted light are shown for each interface (c). Total internal reflection (TIR) within the diamond knife at the diamond/water interface results in light scattering and emission through the knife tip when the incident angle is less than the critical angle (33.3°) for a diamond knife (η = 2.42) immersed in water (η = 1.33) (d). The density of illumination around the knife is estimated using a first-order Monte Carlo simulation (e–f). The amount of scattering is determined by the NA of the light guide. Light intensity is shown as a factor of input intensity using the associated color map. Imaging is performed using time-delayed integation (TDI) as the tissue moves along the 35° bevel at the knife tip. Using TDI increases the SNR and increases the amount of scattered illumination used to construct the final image.

Visualization of the whole-brain vascular data set. The whole downsampled brain is shown (a–b) along with cross-sections composed of ≈ 200 slices (c–f). Scale bars = 2mm. (g) Close-up of an anterior coronal section through the cortex. Features labeled are (1) surface of the lateral ventricles, (2) Pericallosal artery, and (3) the cortical surface with descending microvessels. (h–i) Close-up of a volume reconstruction of the labeled region. Color value indicates estimated vessel radius computed by performing a distance transform on the medial axis.