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使用探针刷新 STORM 的扩展深度 3D 超分辨率成像。

Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM.

机构信息

Department of Chemistry, University of Washington, Seattle, Washington; Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, People's Republic of China.

Department of Chemistry, University of Washington, Seattle, Washington.

出版信息

Biophys J. 2018 Apr 24;114(8):1980-1987. doi: 10.1016/j.bpj.2018.03.023.

Abstract

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.

摘要

用于超分辨率荧光显微镜的单分子定位显微镜方法,如 STORM(随机光学重建显微镜),由于焦平面外的分子发生光漂白,通常仅限于薄的三维(3D)切片(≤600nm)。尽管可以通过在样品内逐渐加深焦点来在光漂白之前对多个焦平面进行成像,但这种方法的图像质量会受到影响,因为可测量的局部定位数量在检测平面之间分配。在这里,我们通过开发一种我们称为探针刷新 STORM(prSTORM)的成像方法来解决固定样品中的这个问题,该方法允许直接用非漂白荧光团替换漂白荧光团。我们通过用 DNA 缀合抗体对样品进行免疫染色,然后使用可以在连续的成像轮次中完全替换的荧光标记 DNA 报告寡核苷酸来读出它们的分布来实现这一点。我们证明 prSTORM 可以在不牺牲任何给定平面上定位密度的情况下,在扩展的深度上获取 3D 图像。我们还表明,prSTORM 可以适应获取具有扩展深度的高质量 3D 多通道图像,这对于使用现有探针方法来说是具有挑战性或不可能实现的。

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