Huang Wei E, Stoecker Kilian, Griffiths Robert, Newbold Lyndsay, Daims Holger, Whiteley Andrew S, Wagner Michael
Biodiversity and Ecosystem Function Group, Molecular Microbial Ecology Section, Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford, OX1 3SR, UK.
Environ Microbiol. 2007 Aug;9(8):1878-89. doi: 10.1111/j.1462-2920.2007.01352.x.
We have coupled fluorescence in situ hybridization (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of (13)C incorporation into microbial cells. Highly resolved Raman confocal spectra were generated for individual cells which were grown in minimal medium where the ratio of (13)C to (12)C content of the sole carbon source was incrementally varied. Cells which were (13)C-labelled through anabolic incorporation of the isotope exhibited key red-shifted spectral peaks, the calculated 'red shift ratio' (RSR) being highly correlated with the (13)C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of Fluos, Cy3 and Cy5-labelled microbial populations at the single cell level. Cellular (13)C-content determinations exhibited good congruence between fresh cells and FISH hybridized cells indicating that spectral peaks, including phenylalanine resonance, which were used to determine (13)C-labelling, were preserved during fixation and hybridization. In order to demonstrate the suitability of this technology for structure-function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseudomonas populations during naphthalene degradation in groundwater microcosms. Raman-FISH extends and complements current technologies such as FISH-microautoradiography and stable isotope probing in that it can be applied at the resolution of single cells in complex communities, is quantitative if suitable calibrations are performed, can be used with stable isotopes and has analysis times of typically 1 min per cell.
我们将荧光原位杂交（FISH）与拉曼显微镜相结合，用于在不依赖培养的情况下同时鉴定微生物细胞并测定其对¹³C的掺入情况。我们为在基础培养基中生长的单个细胞生成了高分辨率的拉曼共聚焦光谱，在该培养基中，唯一碳源的¹³C与¹²C含量之比逐渐变化。通过同位素的同化掺入而被¹³C标记的细胞表现出关键的红移光谱峰，计算出的“红移比”（RSR）与细胞的¹³C含量高度相关。随后，对拉曼仪器和FISH方案进行了优化，以实现对Fluos、Cy3和Cy5标记的微生物群体在单细胞水平上的落射荧光和拉曼成像的联合检测。新鲜细胞与FISH杂交细胞的细胞¹³C含量测定结果显示出良好的一致性，这表明用于确定¹³C标记的光谱峰，包括苯丙氨酸共振峰，在固定和杂交过程中得以保留。为了证明该技术在复杂微生物群落结构-功能分析中的适用性，我们应用拉曼-FISH技术展示了假单胞菌群体在地下水微宇宙中萘降解过程中的重要性。拉曼-FISH技术扩展并补充了当前的技术，如FISH-微放射自显影和稳定同位素探测，因为它可以应用于复杂群落中单个细胞的分辨率水平，若进行适当校准则具有定量功能，可以与稳定同位素一起使用，并且每个细胞的分析时间通常为1分钟。
