Department of Pharmacology School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
Anal Chem. 2020 Apr 21;92(8):5656-5660. doi: 10.1021/acs.analchem.0c00673. Epub 2020 Mar 27.
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 CH vibrational 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.
相干反斯托克斯拉曼散射（CARS）成像广泛用于对细胞和组织内的分子振动进行成像。由于脂质双层含有丰富的 CH 振动键，因此它们是 CARS 成像的潜在分析物。然而，由于其具有薄的结构并且与细胞内的脂质结构紧密相邻，因此鉴定质膜具有挑战性。由于质膜提供了细胞内最突出的不对称位置，因此当使用 CARS 显微镜系统时，具有非荧光和 SFG 特异性的染料 Ap3 标记的质膜的选择性可视化是一种很有前途的技术。在这项研究中，我们使用巨囊泡（GVs）和 N27 大鼠多巴胺能神经细胞，通过基于染料的 SFG 和 CARS 对脂质双层成像的特性进行了仔细比较。结果表明，CARS 成像可用于可视化 GV 和细胞内的整个脂质结构，但不足以识别质膜，而基于染料的 SFG 成像可以实现质膜的识别。此外，我们证明可以将这些独特的特性结合起来，并应用于活细胞内脂质结构（例如质膜下的脂滴）的跟踪。因此，通过基于染料的 SFG 和 CARS 的组合进行多模态多光子成像可以作为一种强大的化学成像工具，用于研究 GV 和活细胞中的脂质双层。
