Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function
Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function
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DOI：
10.1073/pnas.0900245106
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发表时间：
2009-03-03
影响因子：
11.1
通讯作者：
Moerner, W. E.
中科院分区：
文献类型：
--
作者：
Pavani, Sri Rama Prasanna;Thompson, Michael A.;Moerner, W. E.
我们展示了单分子荧光成像超过光学衍射极限的三维宽视场显微镜，显示双螺旋点扩散函数（DH-PSF）。DH-PSF设计具有高且均匀的Fisher信息，并且在成像平面上具有2个主导瓣，其角取向随发射器的轴向(z)位置旋转。厚聚合物样品中的单个荧光分子通过寻找2个DH-PSF瓣的中心，在大景深（2 μ m）内以10至20纳米的精度定位在单个500 ms的采集中。通过使用光激活荧光团，用DH-PSF显微镜对稀疏子集进行重复成像，可以在所有3个维度上提供高浓度分子的超分辨率成像。光学PSF设计与光激活荧光团的数字后处理相结合，为提高超过瑞利衍射极限的3D成像分辨率开辟了道路。
We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 mu m) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.