Adaptive-illumination STED nanoscopy
Adaptive-illumination STED nanoscopy
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DOI：
10.1073/pnas.1708304114
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发表时间：
2017-09-12
影响因子：
11.1
通讯作者：
Hell, Stefan W.
中科院分区：
文献类型：
--
作者：
Heine, Joern;Reuss, Matthias;Hell, Stefan W.
被称为STED/RESOLFT的概念通过两种不同状态之间紧密堆积的分子的光驱动转移来实现超分辨功能，通常是在亚衍射尺度上明确定义的坐标处的非荧光“关闭”状态和荧光“打开”状态。为此，所施加的光强度必须足以保证在所寻求的分辨率下间隔的分子的状态差。因此，在整个成像过程中应用相对高的强度以获得最高的分辨率。在特征相距足够远的区域，分子可以用较低的强度分离，多余的强度只会增加光漂白。在这里，我们介绍DyMIN（动态强度最小值）扫描，概括和扩展早期的概念，救援和MINFIELD，以减少样品曝光。DyMIN的原理是，它只使用尽可能多的开/关开关光，以所需的分辨率成像。可以在亚分辨率邻域内发现荧光团的那些位置处记录荧光。通过在每个像素/体素的采集期间调整强度（以及分辨率），我们将该邻域的大小与正在成像的结构相匹配。DyMIN被示出为在常见生物成像条件下将扫描区域上的STED光的剂量降低至接近20倍，并且对于较稀疏的2D和3D样本降低>100倍。漂白减少可以被转换成相应地更亮的图像，
The concepts called STED/RESOLFT superresolve features by a light-driven transfer of closely packed molecules between two different states, typically a nonfluorescent "off" state and a fluorescent "on" state at well-defined coordinates on subdiffraction scales. For this, the applied light intensity must be sufficient to guarantee the state difference for molecules spaced at the resolution sought. Relatively high intensities have therefore been applied throughout the imaging to obtain the highest resolutions. At regions where features are far enough apart that molecules could be separated with lower intensity, the excess intensity just adds to photobleaching. Here, we introduce DyMIN (standing for Dynamic Intensity Minimum) scanning, generalizing and expanding on earlier concepts of RESCue and MINFIELD to reduce sample exposure. The principle of DyMIN is that it only uses as much on/off-switching light as needed to image at the desired resolution. Fluorescence can be recorded at those positions where fluorophores are found within a subresolution neighborhood. By tuning the intensity (and thus resolution) during the acquisition of each pixel/voxel, we match the size of this neighborhood to the structures being imaged. DyMIN is shown to lower the dose of STED light on the scanned region up to similar to 20-fold under common biological imaging conditions, and >100-fold for sparser 2D and 3D samples. The bleaching reduction can be converted into accordingly brighter images at