Polisetti Sneha, Bible Amber N, Morrell-Falvey Jennifer L, Bohn Paul W
Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
Analyst. 2016 Apr 7;141(7):2175-82. doi: 10.1039/c6an00080k. Epub 2016 Mar 7.
Chemical imaging of plant-bacteria co-cultures makes it possible to characterize bacterial populations and behaviors and their interactions with proximal organisms, under conditions closest to the environment in the rhizosphere. Here Raman micro-spectroscopy and confocal Raman imaging are used as minimally invasive probes to study the rhizosphere bacterial isolate, Pantoea sp. YR343, and its co-culture with model plant Arabidopsis thaliana by combining enhanced Raman spectroscopies with electron microscopy and principal component analysis (PCA). The presence of carotenoid pigments in the wild type Pantoea sp. YR343 was characterized using resonance Raman scattering, which was also used to confirm successful disruption of the crtB gene in an engineered carotenoid mutant strain. Other components of the Pantoea sp. YR343 cells were imaged in the presence of resonantly enhanced pigments using a combination of surface enhanced Raman imaging and PCA. Pantoea sp. YR343 cells decorated with Ag colloid synthesized ex situ gave spectra dominated by carotenoid scattering, whereas colloids synthesized in situ produced spectral signatures characteristic of flavins in the cell membrane. Scanning electron microscopy (SEM) of whole cells and transmission electron microscopy (TEM) images of thinly sliced cross-sections were used to assess structural integrity of the coated cells and to establish the origin of spectral signatures based on the position of Ag nanoparticles in the cells. Raman imaging was also used to characterize senescent green Arabidopsis thaliana plant roots inoculated with Pantoea sp. YR343, and PCA was used to distinguish spectral contributions from plant and bacterial cells, thereby establishing the potential of Raman imaging to visualize the distribution of rhizobacteria on plant roots.
对植物 - 细菌共培养物进行化学成像，能够在最接近根际环境的条件下，对细菌群体及其行为以及它们与邻近生物体的相互作用进行表征。在这里，拉曼显微光谱和共焦拉曼成像被用作微创探针，通过将增强拉曼光谱与电子显微镜和主成分分析（PCA）相结合，来研究根际细菌分离株泛菌属YR343及其与模式植物拟南芥的共培养物。利用共振拉曼散射对野生型泛菌属YR343中类胡萝卜素色素的存在进行了表征，该技术还用于确认工程化类胡萝卜素突变菌株中crtB基因的成功破坏。在存在共振增强色素的情况下，使用表面增强拉曼成像和PCA的组合对泛菌属YR343细胞的其他成分进行成像。用异位合成的银胶体修饰的泛菌属YR343细胞给出的光谱以类胡萝卜素散射为主，而原位合成的胶体产生了细胞膜中黄素的特征光谱特征。使用全细胞扫描电子显微镜（SEM）和薄切片横截面的透射电子显微镜（TEM）图像来评估包被细胞的结构完整性，并根据细胞中银纳米颗粒的位置确定光谱特征的来源。拉曼成像还用于表征接种了泛菌属YR343的衰老绿色拟南芥植物根，PCA用于区分植物和细菌细胞的光谱贡献，从而确定拉曼成像在可视化植物根上根际细菌分布方面的潜力。
