Robust scan synchronized force-fluorescence imaging.

Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells. To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces accurate pixel binning and enables the collection of a scanned image of a sample while applying force to a single point on the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

同步原子力显微镜（AFM）和样品扫描共聚焦荧光显微镜测量被广泛用于获取活细胞中蛋白质动力学的机制和结构方面的见解。然而，由于缺乏一种能同时对原子力显微镜和共聚焦显微镜的压电平台进行同步扫描的可靠技术，很难观察到细胞中荧光蛋白分布因受力而产生的变化。为了应对这一挑战，我们构建了一个集成的原子力显微镜 - 共聚焦荧光显微镜平台，该平台采用了一种同步扫描方法，这种方法消除了压电运动斜坡产生的图像伪影，实现了精确的像素合并，并能够在对样品上的单个点施加力的同时收集样品的扫描图像。作为原理验证，我们使用该仪器在施加机械力时监测活细胞中荧光E - 钙粘蛋白（一种必需的跨膜蛋白）的重新分布。