Li Ang, Ho Bow, Ding Jeak Ling, Lim Chwee Teck
Singapore-MIT Alliance (SMA), National University of Singapore, Kent Ridge, Singapore.
Methods Mol Biol. 2010;618:235-47. doi: 10.1007/978-1-60761-594-1_15.
Atomic force microscopy (AFM) has been extensively used to image the three-dimensional surface morphology of a broad range of biological samples, including Gram-negative bacteria, imaged in the presence of antimicrobial peptides (AMPs). Although this technique provides high molecular resolution, it only requires minimum sample treatment and can even be performed in liquid and at varying temperatures while keeping the bacterial cells viable. In this chapter, we describe an easy, fast, and yet effective method for preparing AMP-treated Gram-negative bacteria samples for AFM imaging. The results obtained using this method show a series of morphological changes of Gram-negative bacteria upon treatment with selected AMPs, thus providing vivid insights into the mechanisms of how AMPs perturb and destroy Gram-negative bacteria in a stepwise manner. Technical details for performing AFM so as to obtain reliable and high-resolution images will also be discussed, together with some possible artifacts and troubleshooting.
原子力显微镜（AFM）已被广泛用于对多种生物样品的三维表面形态进行成像，包括在抗菌肽（AMPs）存在下成像的革兰氏阴性菌。尽管该技术提供了高分子分辨率，但它只需要最少的样品处理，甚至可以在液体中以及不同温度下进行，同时保持细菌细胞的活力。在本章中，我们描述了一种简单、快速且有效的方法，用于制备经AMPs处理的革兰氏阴性菌样品以进行AFM成像。使用该方法获得的结果显示了经选定AMPs处理后革兰氏阴性菌的一系列形态变化，从而为AMPs如何逐步干扰和破坏革兰氏阴性菌的机制提供了生动的见解。还将讨论进行AFM以获得可靠高分辨率图像的技术细节，以及一些可能的伪像和故障排除方法。
