The cytoplasmic membrane is one of the most frequent cell targets of antimicrobial peptides (AMPs) and other biomolecules. Understanding the mechanism of action of AMPs at the molecular level is of utmost importance for designing of new membrane-specific molecules. In particular, the formation of pores, the structure and size of these pores are of great interest and require nanoscale resolution approaches, therefore, biophysical strategies are essential to achieve an understanding of these processes at this scale. In the case of membrane active peptides, pore formation or general membrane disruption is usually the last step before cell death, and so, pore size is generally directly associated to pore structure and stability and loss of cellular homeostasis, implicated in overall peptide activity. Up to date, there has not been a critical review discussing the methods that can be used specifically for estimating the pore dimensions induced by membrane active peptides. In this review we discuss the scope, relevance and popularity of the different biophysical techniques such as liposome leakage experiments, advanced microscopy, neutron or X-ray scattering, electrophysiological techniques and molecular dynamics studies, all of them useful for determining pore structure and dimension.

细胞质膜是抗微生物肽（AMP）和其他生物分子的最常见细胞靶标之一。在设计新的膜特异性分子时，了解AMP在分子水平上的作用机理至关重要。尤其是，孔的形成，这些孔的结构和尺寸引起人们极大的兴趣，并且需要纳米级的分辨率方法，因此，生物物理策略对于在此范围内理解这些过程至关重要。就膜活性肽而言，孔形成或一般的膜破坏通常是细胞死亡前的最后一步，因此，孔径通常与孔结构，细胞稳态和细胞稳态的丧失直接相关，这与总肽活性有关。最新，尚未有针对性的评论来讨论可用于估计膜活性肽诱导的孔尺寸的方法。在这篇综述中，我们讨论了不同生物物理技术的范围，相关性和受欢迎程度，例如脂质体泄漏实验，高级显微镜，中子或X射线散射，电生理技术和分子动力学研究，它们对于确定孔的结构和尺寸都是有用的。