J Phys Chem B. 2018 May 17;122(19):5006-5019. doi: 10.1021/acs.jpcb.8b01537. Epub 2018 May 9.
Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.
和频产生（SFG）振动光谱学可以选择性地检测和分析非中心对称成分在无定形基质中的分布；这一原理已被用于研究植物细胞壁中纤维素的纳米级结构和介观组装。然而，在大面积或大体积上进行平均化处理后的光谱信息无法提供植物中不同细胞的区域特异性或组织特异性信息。本研究通过将宽带 SFG 光谱系统与光学显微镜相结合，展示了空间分辨 SFG 分析和成像。该系统的设计目的是通过单个反射物镜同时辐照窄带 800nm 和宽带可调谐 IR 光束，但从样品表面法向的相反侧入射。所开发的技术用于揭示棉纤维、洋葱表皮等单个细胞壁内纤维素微纤维的不均匀分布，以及拟南芥花序茎和竹子节间不同组织之间的分布。SFG 显微镜可与传统的傅里叶变换红外光谱和共聚焦拉曼显微镜互补，用于其他生物系统的振动光谱成像。
