Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins
Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins
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DOI：
10.1073/pnas.0506010102
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发表时间：
2005-12-06
影响因子：
11.1
通讯作者：
Hell, SW
中科院分区：
文献类型：
--
作者：
Hofmann, M;Eggeling, C;Hell, SW
荧光显微镜在许多科学领域都是不可或缺的，但直到最近，衍射还限制了其基于透镜的变体的分辨率。受激发射导致标记物荧光状态饱和耗尽，衍射屏障已被打破，但这种方法需要 GW/cm(2) 强度的皮秒激光脉冲。在这里，我们展示了在照明强度小八个数量级的情况下超越荧光显微镜中的衍射屏障。亚衍射分辨率是由标记蛋白在荧光激活状态和非激活状态之间的可逆光转换产生的，其中一种转变是通过以零为特征的空间强度分布来完成的。在表征所用标记蛋白 asFP595 的转换动力学后，我们证明了这种 RESOLFT（可逆饱和光学荧光跃迁）类型概念在焦平面中解析 50-100 nm 的当前能力。观察到的分辨率仅受蛋白质的光动力学和零的完美性限制。我们的结果强调了通过技术优化最终在荧光显微镜中实现分子分辨率的潜力。
Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm(2) intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.