Imaging live bacteria at the nanoscale: comparison of immobilisation strategies
Imaging live bacteria at the nanoscale: comparison of immobilisation strategies
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DOI：
10.1101/685024
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发表时间：
2019-07
期刊：
影响因子：
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通讯作者：
Georgina Benn;Alice L. B. Pyne;M. Ryadnov;B. Hoogenboom
中科院分区：
文献类型：
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作者：
Georgina Benn;Alice L. B. Pyne;M. Ryadnov;B. Hoogenboom
原子力显微镜(AFM)提供了一种有效的、无标记的技术，能够以纳米级的精度对生理条件下的活细菌进行成像。然而，原子力显微镜是一种表面扫描技术，其性能的准确性需要有效和可靠地将细菌细胞固定在基质上。在这里，我们比较了各种化学方法，以促进固定化大肠杆菌在玻璃盖片上的有效性，细菌的吸附，生存能力和与相关成像的兼容性通过荧光显微镜。我们使用明胶、聚L赖氨酸、CellTak™和Vectabond®评估表面功能化。我们描述了细菌的固定化、活性和原子力显微镜实验的适用性如何取决于细菌菌株、缓冲条件和表面功能化。我们通过AFM图像演示了这种固定化的使用，这些图像分解了细菌表面的孔蛋白晶格；抗菌肽(抗菌肽B)对细菌细胞膜的局部降解；以及在细菌膜上形成膜攻击复合体。
Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-L-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane.