Integrated Fluorescent Nanoprobe Design for High‐Speed In Vivo Two‐Photon Microscopic Imaging of Deep‐Brain Vasculature in Mice
Minami Takezaki, Ryosuke Kawakami, Shozo Onishi, Yasutaka Suzuki, Jun Kawamata, Takeshi Imamura, Shingo Hadano, Shigeru Watanabe, Yosuke Niko
Abstract
Abstract High‐speed two‐photon microscopy can be used to analyze vascular dynamics in living animals and is essential for the understanding of brain diseases. Recent advances in fluorescent probes/optical systems have allowed successful imaging of the hippocampal vasculature in the deep brain of mice (1 mm from the brain surface) under low‐speed conditions (1–2 fps); however, using high‐speed techniques (>30 fps), observation of the deep‐brain vasculature is still challenging. Here, a new nanoemulsion that encapsulates thousands of red‐emissive pyrene dye molecules while maintaining their high two‐photon brightness [1.5 × 10 2 GM (GM = 10 −50 cm 4 ·s·photon −1 ·molecule −1 ) at 960 nm excitation] and delivers a large amount of such pyrene dyes (65 nmol) into the blood vessels of mice is developed. Remarkably, the nanoprobe is found to exploit the inherent performance of a commonly used Ti:sapphire excitation laser and a sensitive gallium arsenide phosphide nondescanned fluorescence detector to the limit, enabling visualization of the brain vasculature under the cortex region of mice (up to 1.5 mm) under very low‐speed conditions. As a highlight, such a nanoprobe is successfully used to directly observe the blood flow in the hippocampal CA1 region (1.1 mm) through high‐speed resonant scanning (120 fps).