Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus
Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus
复制标题
DOI：
10.1073/pnas.1114444108
复制
发表时间：
2011-11-15
影响因子：
11.1
通讯作者：
Moerner, W. E.
中科院分区：
文献类型：
--
作者：
Lew, Matthew D.;Lee, Steven F.;Moerner, W. E.
最近，单分子成像和光控已经使超分辨率光学显微镜的细胞结构超出阿贝的衍射极限，扩展了活细胞内的结构的非侵入性成像的前沿。然而，活细胞超分辨率成像已经受到挑战，需要相对于其生物背景，如细胞膜的三维（3D）结构的图像。我们已经开发了一种技术，称为超分辨率的功率依赖性主动反射率和点积累的成像在纳米级拓扑结构（SPRAIPAINT），结合成像的细胞内增强的YFP（eYFP）融合（SPRAI）与随机定位的细胞表面（PAINT）图像两个不同的荧光团顺序只有一个激光。使用简单的光诱导的eYFP闪烁和尼罗红到细胞表面上的碰撞通量来实现单分子定位，而不需要任何抗体标记、细胞膜透化或硫醇-氧清除剂系统。在这里，我们展示了活细胞的3D超分辨率成像的新月形，eYFP，细胞骨架荧光蛋白融合，共定位与表面的细菌Caulobacter crescentus使用双螺旋点扩散函数显微镜。细胞内蛋白质结构和细胞表面的三维共定位与超分辨率光学显微镜打开了活细胞中蛋白质相互作用的分析的大门，具有出色的精度（20-40 nm的3D）在一个大的视野（12 × 12 μ m）。
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, livecell 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 x 12 mu m).