Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

Podosomes 是机械敏感的附着/入侵结构，在细胞的基质-粘附界面上形成，并突出到细胞外基质中进行探测和重塑。尽管它们在许多细胞过程中发挥着核心作用，但它们确切的分子结构和功能仍然只被部分了解。我们回顾了足体结构分子尺度成像的最新进展，包括我们新开发的称为 HAWK 的定位显微镜技术，该技术能够实现足体环蛋白的无伪影活细胞超分辨率显微镜，并报告结合荧光定位显微镜 (STORM/ PALM) 和原子力显微镜 (AFM) 在一种设置上，其中定位显微镜提供荧光标记的足体组件的位置和动态，而刚度的空间变化是用 AFM 映射的。对于双色定位显微镜，我们将 iFluor-647（之前已证明无需在成像模式之间更换缓冲液）与光开关蛋白 mEOS3.2 相结合，后者还可实现活细胞成像。