Sandin Joree N, Aryal Surya P, Wilkop Thomas, Richards Christopher I, Grady Martha E
Department of Mechanical Engineering, University of Kentucky.
Department of Chemistry, University of Kentucky.
J Vis Exp. 2020 Aug 11(162). doi: 10.3791/61433.
Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe "pokes" the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.
在过去几十年里，可用于微米和纳米尺度力学表征的技术呈爆发式增长。从扫描电子显微镜和透射电子显微镜的进一步发展，到原子力显微镜的发明，以及荧光成像技术的进步，能够研究微小材料的技术取得了显著进展。Conpokal是一个混成词，它将共聚焦显微镜与原子力显微镜（AFM）结合在一起，其中一个探针“戳”表面。虽然每种技术本身在定性和/或定量图像采集方面都非常有效，但Conpokal提供了通过混合荧光成像和力学表征进行测试的能力。Conpokal专为近乎同时进行共聚焦成像和原子力探测而设计，便于对活的微生物样本进行实验。配对仪器带来的额外洞察力使通过AFM测量的力学性能（如弹性模量、粘附力、表面粗糙度）与通过共聚焦显微镜可观察到的亚细胞成分或活性实现共定位。这项工作提供了一个逐步的方案，用于同时操作激光扫描共聚焦显微镜和原子力显微镜，以实现对同一细胞、同一区域的共聚焦成像和力学表征。
