Integrated Fluorescent Nanoprobe Design for High‐Speed In Vivo Two‐Photon Microscopic Imaging of Deep‐Brain Vasculature in Mice
Integrated Fluorescent Nanoprobe Design for High‐Speed In Vivo Two‐Photon Microscopic Imaging of Deep‐Brain Vasculature in Mice
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DOI：
10.1002/adfm.202010698
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发表时间：
2021-03
影响因子：
19
通讯作者：
Minami Takezaki;R. Kawakami;S. Onishi;Yasutaka Suzuki;J. Kawamata;Takeshi Imamura;Shingo Hadano;Shigeru Watanabe;Y. Niko
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文献类型：
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作者：
Minami Takezaki;R. Kawakami;S. Onishi;Yasutaka Suzuki;J. Kawamata;Takeshi Imamura;Shingo Hadano;Shigeru Watanabe;Y. Niko
高速双光子显微镜可用于分析活体动物的血管动力学，对了解脑部疾病至关重要。荧光探针/光学系统的最新进展已经允许在低速条件下（1 - 2 fps）成功成像小鼠脑深部（距脑表面1mm）的海马血管系统；然而，使用高速技术（bbb30 fps），观察脑深部血管系统仍然具有挑战性。本文开发了一种新的纳米乳液，该纳米乳液封装了数千个红色发射芘染料分子，同时在960 nm激发下保持其高双光子亮度[1.5 × 102 GM (GM = 10−50 cm4·s·光子−1·分子−1)]，并将大量的这种芘染料（65 nmol）输送到小鼠血管中。值得注意的是，该纳米探针充分利用了常用的钛蓝宝石激发激光器和敏感的砷化镓磷化非失扫描荧光探测器的固有性能，能够在极低速条件下可视化小鼠皮层区域（最大1.5 mm）的血管。值得注意的是，该纳米探针成功地通过高速共振扫描（120 fps）直接观察了海马CA1区（1.1 mm）的血流。
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).