Department of Chemistry and Biochemistry, University of Regina, 3737 Wascana Parkway, Regina, SK, S4S 0A2, Canada.
JPK Instruments, JPK Instruments AG, Colditzstr. 34-36, 12099, Berlin, Germany.
Sci Rep. 2018 May 29;8(1):8305. doi: 10.1038/s41598-018-26433-1.
There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.
在将人为化学物质释放到环境中之前，迫切需要评估它们对模型细胞的影响，以帮助预测它们对环境和人类健康的潜在影响。激光扫描共聚焦显微镜（LSCM）和原子力显微镜（AFM）都为细胞生理学提供了丰富的信息。除了确定表面结构外，AFM 在定量成像（QI）模式下还可以使用最小的应用力探测表面生物化学和细胞力学，而 LSCM 则为荧光标记的大分子成像提供了细胞内的窗口。相关的 AFM-LSCM 可提供有关不同细胞特性的互补信息，以全面了解细胞行为。我们提出了一种相关的 AFM-QI-LSCM 测定法，用于对原位活细胞进行实时成像，从而产生有关细胞形态和力学、表面粘附和超微结构以及多个荧光标记大分子实时定位的多路复用数据。为了证明这种方法对不同细胞类型的广泛适用性，我们展示了暴露于 2,4-二氯苯氧基乙酸的模型细菌、真菌和人类细胞的表面性质、内部分子排列和氧化应激的改变。AFM-QI-LSCM 广泛适用于多种细胞类型，可用于评估任何多种污染物单独或组合的影响。
