NIR-II-Excitable Dye-Loaded Nanoemulsions for Two-Photon Microscopy Imaging of Capillary Blood Vessels in the Entire Hippocampal CA1 Region of Living Mice
NIR-II-Excitable Dye-Loaded Nanoemulsions for Two-Photon Microscopy Imaging of Capillary Blood Vessels in the Entire Hippocampal CA1 Region of Living Mice
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DOI：
10.1021/acsami.2c03299
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发表时间：
2022-09-05
影响因子：
9.5
通讯作者：
Niko，Yosuke
中科院分区：
文献类型：
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作者：
Matsuura，Hitomi;Kawakami，Ryosuke;Niko，Yosuke
对于活体双光子荧光显微镜(2PM)成像，能够提高可观察深度和时间分辨率的技术的发展是解决生物和生物医学问题的重要挑战，例如活体动物脑深部(通常是海马区)的血管动力学。通过两种方法实现了改进：一种是光学方法，使用在第二近红外波长区域(NIR-II，1100-1350 nm)振荡的高组织穿透性激发激光；另一种是化学方法，使用具有高双光子亮度的荧光探针(表征为双光子吸收截面σ2和荧光量子产率Φ的乘积)。为了整合这两种方法，我们开发了一种荧光染料，在1100 nm处显示出足够高的σ2Φ值，68个Goeppert-Mayer单位。当使用包裹1000个染料分子的纳米乳剂和1100 nm激光进行2 PM成像时，几乎整个小鼠大脑海马区(表面下约1.1-1.5 mm)的毛细血管都能以30帧的帧速率清晰地显示出来，S-1(平均超过8帧，实际上是3.75帧S-1)。这一可观察到的深度和帧速率比以前关于2 PM成像的报告要高得多。此外，这种纳米乳状液可以显示1.8毫米深的血管，对应于海马齿状回。这些结果突出了将明亮的探测器与NIR-II激光相结合的优势。我们的探头是研究活体动物血管动力学和相关疾病的一种有前途的工具。
For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100–1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ2, and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ2Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1–1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s–1(averaged over eight frames, practically 3.75 frames s–1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.