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In situ probing the interior of single bacterial cells at nanometer scale

机译：纳米级原位探测单个细菌细胞的内部

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摘要

We report a novel approach to probe the interior of single bacterial cells at nanometre resolution by combining focused ion beam (FIB) and atomic force microscopy (AFM). After removing layers of pre-defined thickness in the order of 100 nm on the target bacterial cells with FIB milling, AFM of different modes can be employed to probe the cellular interior under both ambient and aqueous environments. Our initial investigations focused on the surface topology induced by FIB milling and the hydration effects on AFM measurements, followed by assessment of the sample protocols. With fine-tuning of the process parameters, in situ AFM probing beneath the bacterial cell wall was achieved for the first time. We further demonstrate the proposed method by performing a spatial mapping of intracellular elasticity and chemistry of the multi-drug resistant strain Klebsiella pneumoniae cells prior to and after it was exposed to the 'last-line' antibiotic polymyxin B. Our results revealed increased stiffness occurring in both surface and interior regions of the treated cells, suggesting loss of integrity of the outer membrane from polymyxin treatments. In addition, the hydrophobicity measurement using a functionalized AFM tip was able to highlight the evident hydrophobic portion of the cell such as the regions containing cell membrane. We expect that the proposed FIB-AFM platform will help in gaining deeper insights of bacteria-drug interactions to develop potential strategies for combating multi-drug resistance.
机译：我们报告了一种新颖的方法，通过结合聚焦离子束（FIB）和原子力显微镜（AFM），以纳米分辨率探测单个细菌细胞的内部。在通过FIB研磨去除目标细菌细胞上预定厚度为100 nm的层之后，可以采用不同模式的AFM来探测环境和水环境下的细胞内部。我们最初的研究集中在FIB铣削引起的表面拓扑结构以及水化作用对AFM测量的影响，然后评估样品方案。通过对工艺参数的微调，首次实现了细菌细胞壁下方的原位AFM探测。我们通过对多重耐药菌株肺炎克雷伯菌肺炎细胞暴露于“最后一道”抗生素多粘菌素B之前和之后进行细胞内弹性和化学性质的空间作图，进一步证明了所提出的方法。我们的结果表明，刚度增加了在处理过的细胞的表面和内部区域中，这表明多粘菌素处理使外膜的完整性丧失。此外，使用功能化AFM尖端进行的疏水性测量能够突出显示细胞的明显疏水部分，例如包含细胞膜的区域。我们希望提议的FIB-AFM平台将有助于获得细菌与药物相互作用的更深刻见解，从而开发出对抗多种药物耐药性的潜在策略。

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