Direct stochastic optical reconstruction microscopy with standard fluorescent probes
Direct stochastic optical reconstruction microscopy with standard fluorescent probes
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DOI：
10.1038/nprot.2011.336
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发表时间：
2011-06-01
期刊：
影响因子：
14.8
通讯作者：
Sauer, Markus
中科院分区：
文献类型：
--
作者：
van de Linde, Sebastian;Loeschberger, Anna;Sauer, Markus
直接随机光学重建显微镜（dSTORM）使用传统的荧光探针，如标记抗体或化学标签，进行亚衍射分辨率荧光成像，横向分辨率类似于20纳米。与具有光激活荧光蛋白的光激活定位显微镜（PALM）相比，dSTORM实验从明亮的荧光样品开始，其中荧光团必须转移到稳定和可逆的OFF状态。在足够低的光强度照射后，关闭状态的寿命范围为100毫秒至几秒钟，以确保最小的光破坏。在第二激光波长照射下自发或光诱导，荧光团的稀疏子集被重新激活并精确确定其位置。重复的激活、定位和去激活允许重构图像中空间未解析结构的时间分离。在这里，我们提出了一个一步一步的协议dSTORM成像固定细胞和活细胞在宽视场荧光显微镜上，标准荧光探针特别聚焦于光诱导精细调整的比例的荧光团驻留在on和OFF状态。此外，我们还讨论了标记策略、获取参数以及时间和空间分辨率。数据采集和数据处理的最终步骤可以在几秒到几分钟内完成。
Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of similar to 20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.