Multimodal Multiphoton Imaging of the Lipid Bilayer by Dye-Based Sum-Frequency Generation and Coherent Anti-Stokes Raman Scattering
Multimodal Multiphoton Imaging of the Lipid Bilayer by Dye-Based Sum-Frequency Generation and Coherent Anti-Stokes Raman Scattering
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通过基于染料的和频生成和相干反斯托克斯拉曼散射对脂质双层进行多模态多光子成像
DOI：
10.1021/acs.analchem.0c00673
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发表时间：
2020
影响因子：
7.4
通讯作者：
Nuriya Mutsuo
中科院分区：
文献类型：
--
作者：
Mizuguchi Takaha;Momotake Atsuya;Hishida Mafumi;Yasui Masato;Yamamoto Yasuhiko;Saiki Toshiharu;Nuriya Mutsuo
相干反斯托克斯拉曼散射（汽车）成像被广泛用于对细胞和组织内的分子振动进行成像。脂质双分子层由于其丰富的CH2振动键而成为汽车成像的潜在分析物。然而，识别质膜是具有挑战性的，因为它具有薄的结构，并且与细胞内的脂质结构紧密并列。由于质膜提供了最突出的不对称位置内的细胞，方向敏感的和频产生（SFG）成像是一种有前途的技术，选择性可视化的质膜标记的非荧光和SFG特异性染料，Ap3，当使用汽车显微镜系统。在这项研究中，我们密切比较了基于染料的SFG和汽车使用巨大的囊泡（GVs）和N27大鼠多巴胺能神经细胞的脂质双层成像的特点。因此，我们表明汽车成像可以用于可视化GV和细胞内的整个脂质结构，但不足以识别质膜，而这可以使用基于染料的SFG成像来实现。此外，我们证明，这些独特的性能可以结合起来，并适用于活细胞跟踪细胞内的脂质结构，如脂滴下质膜。因此，通过基于染料的SFG和汽车的组合的多模态多光子成像可以作为一个强大的化学成像工具来研究GVs和活细胞中的脂质双层。
Coherent anti-Stokes Raman scattering (CARS) imaging is widely used for imaging molecular vibrations inside cells and tissues. Lipid bilayers are potential analytes for CARS imaging due to their abundant CH2vibrational bonds. However, identifying the plasma membrane is challenging since it possesses a thin structure and is closely apposed to lipid structures inside the cells. Since the plasma membrane provides the most prominent asymmetric location within cells, orientation sensitive sum-frequency generation (SFG) imaging is a promising technique for selective visualization of the plasma membrane labeled by a nonfluorescent and SFG-specific dye, Ap3, when using a CARS microscope system. In this study, we closely compare the characteristics of lipid bilayer imaging by dye-based SFG and CARS using giant vesicles (GVs) and N27 rat dopaminergic neural cells. As a result, we show that CARS imaging can be exploited for the visualization of whole lipid structures inside GVs and cells but is insufficient for identification of the plasma membrane, which instead can be achieved using dye-based SFG imaging. In addition, we demonstrate that these unique properties can be combined and applied to the live-cell tracking of intracellular lipid structures such as lipid droplets beneath the plasma membrane. Thus, multimodal multiphoton imaging through a combination of dye-based SFG and CARS can serve as a powerful chemical imaging tool to investigate lipid bilayers in GVs and living cells.