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Strong cytoskeleton activity on millisecond timescales upon particle binding revealed by ROCS microscopy

机译:ROCS显微镜显示颗粒结合后毫秒时间粒子骨骼活性的强烈细胞骨架活性

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Abstract Cells change their shape within seconds, cellular protrusions even on subsecond timescales enabling various responses to stimuli of approaching bacteria, viruses or pharmaceutical drugs. Typical response patterns are governed by a complex reorganization of the actin cortex, where single filaments and molecules act on even faster timescales. These dynamics have remained mostly invisible due to a superposition of slow and fast motions, but also due to a lack of adequate imaging technology. Whereas fluorescence techniques require too long integration times, novel coherent techniques such as ROCS microscopy can achieve sufficiently high spatiotemporal resolution. ROCS uses rotating back‐scattered laser light from cellular structures and generates a consistently high image contrast at 150?nm resolution and frame rates of 100?Hz—without fluorescence or bleaching. Here, we present an extension of ROCS microscopy that exploits the principles of dynamic light scattering for precise localization, visualization and quantification of the cytoskeleton activity of mouse macrophages. The locally observed structural reorganization processes, encoded by dynamic speckle patterns, occur upon distinct mechanical stimuli, such as soft contacts with optically trapped beads. We find that a substantial amount of the near‐membrane cytoskeleton activity takes place on millisecond timescales, which is much faster than reported ever before.
机译:摘要细胞在几秒钟内改变它们的形状,即使在亚副计时的细胞突起,也能够各种对接近细菌,病毒或药物药物的刺激的各种反应。典型的响应模式受肌动蛋白皮质的复杂重组来控制,其中单细丝和分子在甚至更快的时间表上起作用。由于慢速和快速运动的叠加,这些动态仍然是不可见的,而且由于缺乏足够的成像技术。虽然荧光技术需要太长的整合时间,但是新颖的相干技术,例如Rocs显微镜可达到足够高的时空分辨率。 ROCS使用来自蜂窝结构的旋转背散射的激光,并在150Ω分辨率和100μl帧速率下产生始终如一的高图像对比度 - 没有荧光或漂白。在这里,我们展示了Rocs显微镜的延伸,该参数用于利用动态光散射的原理,以获得小鼠巨噬细胞的细胞骨架活性的精确定位,可视化和定量。通过动态散斑图案编码的本地观察到的结构重组过程发生在不同的机械刺激之上,例如与光学捕获的珠子的软接触。我们发现大量的近膜细胞骨架活性在毫秒的时间表上进行,这比以前的报告速度快得多。

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