Imaging single-cell blood flow in the smallest to largest vessels in the living retina

Tissue light scatter limits the visualization of the microvascular network deep inside the living mammal. The transparency of the mammalian eye provides a noninvasive view of the microvessels of the retina, a part of the central nervous system. Despite its clarity, imperfections in the optics of the eye blur microscopic retinal capillaries, and single blood cells flowing within. This limits early evaluation of microvascular diseases that originate in capillaries. To break this barrier, we use 15 kHz adaptive optics imaging to noninvasively measure single-cell blood flow, in one of the most widely used research animals: the C57BL/6J mouse. Measured flow ranged four orders of magnitude (0.0002-1.55 µL min^-1) across the full spectrum of retinal vessel diameters (3.2-45.8 µm), without requiring surgery or contrast dye. Here, we describe the ultrafast imaging, analysis pipeline and automated measurement of millions of blood cell speeds.