The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer-range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.

中文翻译：

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映射细胞力学的异质性的多谐波原子力显微镜。

机械生物学的目的是了解活细胞的物理特性变化与正常生理学和疾病之间的联系。这需要在单个单元格内具有适当的空间和时间分辨率的机械测量。常规的原子力显微镜（AFM）方法可逐点获取力曲线，用于绘制细胞的异质力学性能。但是，生成的地图获取时间比研究许多动态细胞过程所需的时间长得多。使用共振微悬臂梁的动态AFM（dAFM）方法与高速，高分辨率扫描兼容；但是，它们不能直接获取力曲线，并且需要将有限数量的仪器可观测值转换为局部机械特性图。我们最近开发了一种技术，该技术允许配备有直接悬臂激励的商业AFM系统定量绘制活细胞的粘弹性。可以在几个宽间隔的频率下获得这些特性，这些频率具有纳米范围的空间分辨率和快速的图像采集时间（数十秒）。在这里，我们描述了定量映射的详细步骤，包括样品制备，AFM校准和数据分析。该协议可应用于不同的生物样品，包括细胞和病毒。对于有经验的AFM用户，应该能够轻松实现从dAFM成像到定量标测的过渡，他们将能够在30分钟内建立协议。