Valley Christopher C, Liu Sheng, Lidke Diane S, Lidke Keith A
Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico, United States of America.
Department of Physics & Astronomy, University of New Mexico, Albuquerque, New Mexico, United States of America.
PLoS One. 2015 Apr 10;10(4):e0123941. doi: 10.1371/journal.pone.0123941. eCollection 2015.
Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.
荧光超分辨率（SR）显微镜，即荧光纳米显微镜，可提供细胞结构的纳米级细节，并能在分子水平上对生物过程进行成像。特定的SR成像方法，如基于定位的成像，依赖于荧光团在开启（荧光）和关闭（暗）状态之间的随机转换。使用多色成像对多个细胞结构进行成像很复杂，并且受到各种有机染料不同特性的限制，包括它们的荧光状态占空比、每次转换事件的光子数、不可逆光漂白前的荧光循环次数以及对缓冲条件的总体敏感性。此外，多色成像需要考虑多个光路或色差，这可能导致在纳米尺度上很重要的差异像差。在这里，我们报告了一种顺序标记和成像的方法，该方法允许使用单个荧光团对多个目标进行SR成像，图像之间的串扰可忽略不计。通过利用明场图像相关性在x-y成像平面中以约10 nm的叠加精度配准和叠加多个图像采集，我们利用了AlexaFluor647在直接随机光学重建显微镜（dSTORM）中的最佳特性来对四种不同的细胞蛋白进行成像。我们还可视化了添加表皮生长因子（EGF）后表皮生长因子受体和网格蛋白共定位的变化，这些变化与网格蛋白介导的内吞作用一致。这些结果首次证明了使用直接随机重建显微镜（dSTORM）进行顺序SR（s-SR）成像，并且这种顺序成像方法可应用于任何超分辨率技术。
