Over the past five years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool set capable of imaging the surfaces of biological samples ranging from single receptors to membranes and tissues. One of these approaches, force-distance curve-based AFM (FD-based AFM), uses a probing tip functionalized with a ligand to image living cells at high-resolution and simultaneously localize and characterize specific ligand-receptor binding events. Analyzing data from FD-based AFM experiments using appropriate probabilistic models allows quantification of the kinetic and thermodynamic parameters that describe the free-energy landscape of the ligand-receptor bond. We have recently developed an FD-based AFM approach to quantify the binding events of single enveloped viruses to surface receptors of living animal cells while simultaneously observing them by fluorescence microscopy. This approach has provided insights into the early stages of the interaction between a virus and a cell. Applied to a model virus, we probed the specific interaction with cells expressing viral cognate receptors and measured the affinity of the interaction. Furthermore, we observed that the virus rapidly established specific multivalent interactions and found that each bond formed in sequence strengthened the attachment of the virus to the cell. Here we describe detailed procedures for probing the specific interactions of viruses with living cells; these procedures cover tip preparation, cell sample preparation, step-by-step FD-based AFM imaging and data analysis. Experienced microscopists should be able to master the entire set of protocols in 1 month.

中文翻译：

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共焦和原子力显微镜相结合，以定量单病毒结合到哺乳动物细胞表面。

在过去的五年中，基于原子力显微镜（AFM）的方法已发展成为功能强大的多参数工具集，能够对从单个受体到膜和组织的生物样品的表面进行成像。这些方法之一是基于力距离曲线的AFM（基于FD的AFM），它使用经配体功能化的探针尖端以高分辨率对活细胞成像，并同时定位和表征特定的配体-受体结合事件。使用适当的概率模型分析来自基于FD的AFM实验的数据，可以量化描述配体-受体键的自由能态的动力学和热力学参数。我们最近开发了一种基于FD的AFM方法，用于量化单个包膜病毒与活体动物细胞表面受体的结合事件，同时通过荧光显微镜观察它们。这种方法提供了对病毒和细胞之间相互作用的早期阶段的见识。应用于模型病毒，我们探讨了与表达病毒同源受体的细胞的特异性相互作用，并测量了相互作用的亲和力。此外，我们观察到该病毒迅速建立起特定的多价相互作用，并发现按顺序形成的每个键都增强了该病毒与细胞的附着。在这里，我们描述了探测病毒与活细胞之间特定相互作用的详细程序。这些步骤包括针尖制备，细胞样品制备，基于FD的分步式AFM成像和数据分析。经验丰富的显微镜专家应该能够在1个月内掌握整套方案。