Coordinate-targeted fluorescence nanoscopy with multiple off states
Coordinate-targeted fluorescence nanoscopy with multiple off states
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DOI：
10.1038/nphoton.2015.266
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发表时间：
2016-02-01
期刊：
影响因子：
35
通讯作者：
Hell, Stefan W.
中科院分区：
文献类型：
--
作者：
Danzl, Johann G.;Sidenstein, Sven C.;Hell, Stefan W.
远场超分辨率荧光显微镜通过在检测前将荧光团以不同的状态(通常是开和关)进行短暂的转换，来识别比衍射势垒更近的荧光团。在坐标定向超分辨率变体中，例如受激发射耗尽(STED)显微镜，这种状态差异是由光学图案的强度最小值和最大值造成的，导致所有荧光团例如除了在最小值处之外都处于关闭状态。虽然开启态的强空间限制可以实现高分辨率，但它也使荧光团在最大值时受到过量强度和状态周期的影响。在这里，我们通过驱动荧光团进入第二关断状态来解决这些问题，该状态对过量光是惰性的。通过使用可逆切换的荧光蛋白作为标记，我们的方法减少了漂白，并提高了活细胞STED显微镜的分辨率和对比度。使用两个或更多的跃迁到OFF状态是一个有用的策略，以增加坐标目标超分辨率显微镜的能力。
Far-field super-resolution fluorescence microscopy discerns fluorophores residing closer than the diffraction barrier by briefly transferring them in different (typically ON and OFF) states before detection. In coordinate-targeted super-resolution variants, such as stimulated emission depletion (STED) microscopy, this state difference is created by the intensity minima and maxima of an optical pattern, causing all fluorophores to assume the off state, for instance, except at the minima. Although strong spatial confinement of the on state enables high resolution, it also subjects the fluorophores to excess intensities and state cycles at the maxima. Here, we address these issues by driving the fluorophores into a second off state that is inert to the excess light. By using reversibly switchable fluorescent proteins as labels, our approach reduces bleaching and enhances resolution and contrast in live-cell STED microscopy. Using two or more transitions to off states is a useful strategy for augmenting the power of coordinate-targeted super-resolution microscopy.