PDF(Pubmed)
Abstract：
We previously reported a single-cell adhesion micro tensile tester (SCAμTT) fabricated from IP-S photoresin with two-photon polymerization (TPP) for investigating the mechanics of a single cell-cell junction under defined tensile loading. A major limitation of the platform is the autofluorescence of IP-S, the photoresin for TPP fabrication, which significantly increases background signal and makes fluorescent imaging of stretched cells difficult. In this study, we report the design and fabrication of a new SCAμTT platform that mitigates autofluorescence and demonstrate its capability in imaging a single cell pair as its mutual junction is stretched. By employing a two-material design using IP-S and IP-Visio, a photoresin with reduced autofluorescence, we show a significant reduction in autofluorescence of the platform. Further, by integrating apertures onto the substrate with a gold coating, the influence of autofluorescence on imaging is almost completely mitigated. With this new platform, we demonstrate the ability to image a pair of epithelial cells as they are stretched up to 250% strain, allowing us to observe junction rupture and F-actin retraction while simultaneously recording the accumulation of over 800 kPa of stress in the junction. The platform and methodology presented here can potentially enable detailed investigation of the mechanics of and mechanotransduction in cell-cell junctions and improve the design of other TPP platforms in mechanobiology applications.
摘要：
我们先前报道了一种由IP-S光敏树脂制成的单细胞粘附微拉伸测试仪（SCAμTT），具有双光子聚合（TPP），用于研究在定义的拉伸载荷下单细胞-细胞连接的力学。该平台的一个主要限制是IP-S的自发荧光，用于TPP制造的光敏树脂，这显著增加了背景信号，并使拉伸细胞的荧光成像变得困难。在这项研究中,我们报告了一种新的SCAμTT平台的设计和制造，该平台减轻了自发荧光，并证明了其在单个细胞对相互连接被拉伸时成像的能力。通过采用使用IP-S和IP-Visio的双材料设计，一种自发荧光减少的光敏树脂，我们显示平台的自发荧光显着减少。Further,通过将孔集成到具有金涂层的基板上，自发荧光对成像的影响几乎完全减轻。有了这个新平台,我们展示了成像一对上皮细胞的能力，因为它们被拉伸到250%的应变，使我们能够观察接头破裂和F-肌动蛋白收缩，同时记录接头中超过800kPa应力的积累。此处介绍的平台和方法可以潜在地实现对细胞-细胞连接中的力学和机械转导的详细研究，并改善机械生物学应用中其他TPP平台的设计。
