Integrated Fluorescent Nanoprobe Design for High-Speed In Vivo Two-Photon Microscopic Imaging of Deep-Brain Vasculature in Mice

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 × 10² GM (GM = 10⁻⁵⁰ cm⁴·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).