黄藤细胞壁微纤丝取向偏振光拉曼光谱研究

采用532 nm共聚焦显微拉曼光谱技术原位状态下研究了黄藤藤茎纤维及导管细胞壁中纤维素微纤丝空间取向差异。在高数值孔径（NA=1.25）物镜测试条件下，C-H伸缩振动（2 771~3 000 cm-1）特征峰峰面积拉曼成像成功的区分出细胞角隅、复合胞间层以及次生壁。进一步发现纤维细胞次生壁呈宽窄交替的同心层状结构，而导管次生壁无明显的分层结构。采用平行于细胞径向壁的拉曼偏振激光进行光谱成像发现纤维细胞次生壁窄层纤维素C-O-C（1 097 cm-1）拉曼信号强度明显高于宽层，即窄层中微纤丝取向更加平行于入射激光偏振方向，与细胞轴夹角更大，而导管次生壁中微纤丝取向较为均一。细胞壁不同形态区域拉曼光谱分析发现纤维素C-O-C特征峰以及CH和CH₂特征峰的拉曼信号强度与入射激光的偏振方向存在明显的相关性。当入射偏振激光的电矢量方向从平行变化到垂直于微纤丝方向时，其糖苷键C-O-C非对称伸缩振动信号减弱，而CH和CH₂的取向在与入射偏振激光的电矢量方向垂直时，其拉曼信号强度相较于平行状态略微降低，表明纤维素特征峰中的糖苷键C-O-C的非对称伸缩振动比CH和CH₂伸缩振动对拉曼偏振光的方向改变更为敏感。比较纤维细胞宽层与窄层的拉曼光谱发现径向次生壁窄层1 097 cm-1处拉曼信号强度明显高于弦向次生壁窄层，而径向次生壁宽层的2 897 cm-1处拉曼信号强度低于弦向次生壁宽层。拉曼特征峰比值（I_{1 095}/I_{2 897}）可用来定性研究细胞壁微纤丝角，结果发现这一比值在导管次生壁、纤维细胞窄层和纤维细胞宽层中分别为1.32~1.10，0.92~0.55和0.42~0.33，表明导管次生壁具有最大的微纤丝角，纤维细胞窄层次之，宽层最小。该研究为解析藤材细胞壁骨架空间结构、化学成分分布以及微力学特性提供了新型的分析手段和重要的理论指导。

Study on Microfibrils Orientation in Daemonorops jenkinsiana Cell Wall by Polarized Laser Raman Spectroscopy

The variation in the microfibrils orientation of the Daemonorops jenkinsiana multilayered fiber and vessel was in-situ studied by polarized laser Raman spectroscopy with 532 nm exciting laser and a high-NA objective lens (1.25). Raman imaging obtained by integrating over the band range from 2 771 to 3 000 cm-1 displayed the morphologically distinct cell wall regions, including cell corner middle lamella, compound middle lamella and secondary wall. Furthermore, the fiber secondary wall displayed a concentric structure with alternating broad and narrow layers, while vessels had no obvious layering structure. Higher Raman C-O-C band (1 097 cm-1) intensity was visualized within the narrow layer of fiber wall from polarized laser Raman images, indicating the microfibrilsin these regions were more parallel to the incident laser electric vector and more perpendicular to the cell axis. The microfibrils orientation in vessel was uniform with in its secondary wall. Raman spectral analysis of different morphological areas of cell wall evidenced the band intensities of the C-O-C, CH and CH₂ modes had a significant correlation with the polarization direction of incident light. When the orientation of cellulose microfibrils changed from parallel in respect to the fiber axis to with a high angle, the C-O-C signal reduced obviously. By comparison, the signal of CH and CH₂ displayed slight decrease when changing the direction of incident laser, indicating the more sensitive nature of C-O-C vibration modes to polarization direction of incident light. Comparing the Raman spectra extracted from the narrow (Nl-R), broad layer (Bl-R) of fiber radial wall, as well as narrow (Nl-T), broad layer (Bl-T) of fiber tangential wall, it was found that the Nl-R had higher 1 097 cm-1 band intensity, while Bl-T displayed higher 2 897 cm-1 band intensity. Raman band ratio (I_{1 095}/I_{2 897}) can be used to predict microfibrils angle (MFA) in different cell wall types, qualitatively. The results showed that the ratio was highest in vessel secondary wall (1.32~1.10), followed by narrow layer of fiber secondary wall (0.92~0.55), and lowest in the broad layer of fiber secondary wall (0.42~0.33), indicating the highest MFA in the vessel secondary wall. This study provided a novel method and important theoretical guidance for the investigation on cell wall architecture, chemical composition distribution and micromechanics.