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Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

机译：新月形活细菌中胞内蛋白超结构和细胞表面的三维超分辨率共定位

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Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe’s diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20–40 nm in 3D) over a large field of view (12 × 12 μm).
机译：最近，单分子成像和光控技术已使细胞结构的超高分辨率光学显微镜技术超出了阿贝的衍射极限，从而扩展了活细胞内结构的非侵入性成像的前沿领域。但是，活细胞超分辨率成像已面临着需要对三维（3D）结构相对于其生物学环境（例如细胞膜）成像的挑战。我们已经开发了一种技术，该技术被称为功率依赖性主动间歇性和点累积的超分辨率，用于纳米尺度地形学（SPRAIPAINT）中的成像，该技术将细胞内增强型YFP（eYFP）融合物（SPRAI）的成像与细胞表面随机定位（PAINT）结合在一起仅用一个激光器连续成像两个不同的荧光团。简单的光诱导的eYFP闪烁和尼罗红在细胞表面的碰撞通量被用于实现单分子定位，而无需任何抗体标记，细胞膜通透性或硫醇-氧清除剂系统。在这里，我们演示了使用双螺旋点扩散功能显微镜，Crescentin-eYFP（一种细胞骨架荧光蛋白融合物）的活细胞3D超分辨率成像，与细菌Caulobacter crescentus的表面共定位。利用超高分辨率光学显微镜对细胞内蛋白质结构和细胞表面进行三维共定位，为在宽视场（12×12μm）中以极好的精度（3D，20-40 nm）分析活细胞中的蛋白质相互作用打开了一扇门）。

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