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首页> 外文会议>Conference on single molecule spectroscopy and imaging >Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable Enhanced Yellow Fluorescent Protein
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Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable Enhanced Yellow Fluorescent Protein

机译：使用光学性吸引增强的黄色荧光蛋白在活细胞瘤细胞中的超级化成像

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Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized singlemolecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10 -6 , on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.
机译：近来，光活化和光控分别用来控制单分子荧光标记和超出光学衍射极限（例如，PALM，FPALM和STORM）细胞结构产生图像。虽然以前的活细胞研究上的复杂的光激活荧光蛋白依赖，我们目前的工作表明，超分辨率成像可与融合进行，以常用的荧光蛋白EYFP。而不是被光活化，但是，EYFP可以具有明显的光漂白后紫色光激活。在初始成像之后每个周期中，只有一个子集的稀疏的荧光团被重新激活和局部的，并且最终的图像然后从测得的单分子的位置产生。因为这些方法是基于成像纳米尺寸singlemolecule发射器和在使用主动控制机构，以产生稀疏子合奏，建议的短语“单分子主动控制显微镜”（SMACM），作为一个包容术语这一总体成像策略。在本文中，我们讨论从生理上产生的限制强加在荧光浓度上边界采用暗延时周期，允许单分子运动填写丝状结构，而在最本地化每个发射器每分辨率一旦增加有效标记浓度 - 有限的地方。我们的细菌肌动蛋白的蛋白质MREB的图像细胞周期依赖性上层建筑活柄杆菌新月细胞首次亚40纳米的分辨率。此外，我们量化EYFP的再活化的量子产率，并发现这是1.6×10 -6，在同水准与像Dronpa常规光开关的荧光蛋白。这些研究表明，EYFP是在细菌细胞中的细胞内结构的体内超分辨率成像的有用发射器。

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