Experimental validation of atomic force microscopy-based cell elasticity measurements
Experimental validation of atomic force microscopy-based cell elasticity measurements
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DOI：
10.1088/0957-4484/22/34/345102
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发表时间：
2011-08-26
期刊：
影响因子：
3.5
通讯作者：
Charras, G. T.
中科院分区：
文献类型：
--
作者：
Harris, Andrew R.;Charras, G. T.
原子力显微镜（AFM）被广泛用于测量活细胞的弹性，其产生的值范围为100 Pa至100 kPa，远大于使用微珠跟踪微流变学或微量吸液管抽吸获得的值（100-500 Pa）。AFM弹性测量似乎依赖于尖端几何形状，金字塔形尖端产生的弹性比球形尖端大2-3倍，这种效果通常归因于球形尖端的较大接触面积。在AFM弹性测量中，使用接触力学模型分析实验力-压痕曲线，该模型从尖端几何形状和压痕深度推断尖端-细胞接触面积。这些假设的有效性从未得到证实。在这里，我们利用结合AFM共聚焦显微镜的上皮细胞表达GFP标记的膜标记直接表征压痕的几何形状和测量压痕深度。与来自AFM力压痕曲线的数据比较表明，球形尖端的实验测量的接触面积与预测值一致，而金字塔尖，接触面积可以被严重低估，在大于类似于0.2 nN的力，导致大于两倍的弹性高估。这些数据表明，在文献中报道的绝对细胞弹性的重新检查可能是必要的，我们建议的指导方针，以避免弹性测量的文物引入的外来骨水泥细胞接触。
Atomic force microscopy (AFM) is widely used for measuring the elasticity of living cells yielding values ranging from 100 Pa to 100 kPa, much larger than those obtained using bead-tracking microrheology or micropipette aspiration (100-500 Pa). AFM elasticity measurements appear dependent on tip geometry with pyramidal tips yielding elasticities 2-3 fold larger than spherical tips, an effect generally attributed to the larger contact area of spherical tips. In AFM elasticity measurements, experimental force-indentation curves are analyzed using contact mechanics models that infer the tip-cell contact area from the tip geometry and indentation depth. The validity of these assumptions has never been verified. Here we utilize combined AFM-confocal microscopy of epithelial cells expressing a GFP-tagged membrane marker to directly characterize the indentation geometry and measure the indentation depth. Comparison with data derived from AFM force-indentation curves showed that the experimentally measured contact area for spherical tips agrees well with predicted values, whereas for pyramidal tips, the contact area can be grossly underestimated at forces larger than similar to 0.2 nN leading to a greater than two-fold overestimation of elasticity. These data suggest that a re-examination of absolute cellular elasticities reported in the literature may be necessary and we suggest guidelines for avoiding elasticity measurement artefacts introduced by extraneous cantilever-cell contact.