NIR-II-Excitable Dye-Loaded Nanoemulsions for Two-Photon Microscopy Imaging of Capillary Blood Vessels in the Entire Hippocampal CA1 Region of Living Mice
- Hitomi MatsuuraHitomi MatsuuraResearch and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University, 2-5-1, Akebono-cho, Kochi-shi, Kochi 780-8520, JapanTOSA Innovative Human Development Programs, Kochi University, 2-5-1, Akebono-cho, Kochi-shi, Kochi 780-8520, JapanMore by Hitomi Matsuura
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- Ryosuke KawakamiRyosuke KawakamiDepartment of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, JapanMore by Ryosuke Kawakami
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- Maki IsoeMaki IsoeResearch and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University, 2-5-1, Akebono-cho, Kochi-shi, Kochi 780-8520, JapanMore by Maki Isoe
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- Masaharu HoshiharaMasaharu HoshiharaGraduate School of Sciences and Technology for Innovation, Yamaguchi University, 1677-1, Yoshida, Yamaguchi-shi, Yamaguchi 753-8512, JapanMore by Masaharu Hoshihara
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- Yuya MinamiYuya MinamiGraduate School of Sciences and Technology for Innovation, Yamaguchi University, 1677-1, Yoshida, Yamaguchi-shi, Yamaguchi 753-8512, JapanMore by Yuya Minami
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- Kazuki YatsuzukaKazuki YatsuzukaDepartment of Dermatology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, JapanMore by Kazuki Yatsuzuka
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- Teruko TsudaTeruko TsudaDepartment of Dermatology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, JapanMore by Teruko Tsuda
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- Masamoto MurakamiMasamoto MurakamiDepartment of Dermatology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, JapanMore by Masamoto Murakami
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- Yasutaka SuzukiYasutaka SuzukiGraduate School of Sciences and Technology for Innovation, Yamaguchi University, 1677-1, Yoshida, Yamaguchi-shi, Yamaguchi 753-8512, JapanMore by Yasutaka Suzuki
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- Jun KawamataJun KawamataGraduate School of Sciences and Technology for Innovation, Yamaguchi University, 1677-1, Yoshida, Yamaguchi-shi, Yamaguchi 753-8512, JapanMore by Jun Kawamata
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- Takeshi ImamuraTakeshi ImamuraDepartment of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, JapanMore by Takeshi Imamura
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- Shingo HadanoShingo HadanoResearch and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University, 2-5-1, Akebono-cho, Kochi-shi, Kochi 780-8520, JapanMore by Shingo Hadano
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- Shigeru WatanabeShigeru WatanabeResearch and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University, 2-5-1, Akebono-cho, Kochi-shi, Kochi 780-8520, JapanMore by Shigeru Watanabe
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- Yosuke Niko*Yosuke Niko*Email: [email protected]Research and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University, 2-5-1, Akebono-cho, Kochi-shi, Kochi 780-8520, JapanMore by Yosuke Niko
Abstract
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.
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