Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution
Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution
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DOI：
10.1073/pnas.0406877102
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发表时间：
2005-09-13
影响因子：
11.1
通讯作者：
Gustafsson, MGL
中科院分区：
文献类型：
--
作者：
Gustafsson, MGL
与众所周知的衍射极限相反，荧光显微镜原则上能够实现无限的分辨率。必要的元素是空间结构化的照明光和荧光发射率对照明强度的非线性依赖性。作为这一概念的一个例子，本文实验演示饱和结构照明显微镜，最近提出的方法，其中的非线性激发态的饱和产生。这种方法可以用在一个简单的，宽视场（非扫描）显微镜，只使用一个单一的，廉价的激光，并不需要不寻常的荧光团的物理性质。实际分辨能力由信噪比决定，而信噪比又受到光漂白的限制。实验结果表明，在足够明亮和光稳定的样品上，< 50 nm的2D点分辨率是可能的。
Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of < 50 nm is possible on sufficiently bright and photostable samples.