Pavani Sri Rama Prasanna, Thompson Michael A, Biteen Julie S, Lord Samuel J, Liu Na, Twieg Robert J, Piestun Rafael, Moerner W E
Department of Electrical and Computer Engineering, University of Colorado, Boulder, CO 80309, USA.
Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):2995-9. doi: 10.1073/pnas.0900245106. Epub 2009 Feb 11.
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 microm) 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.
我们使用一种具有双螺旋点扩散函数（DH - PSF）的宽场显微镜，在三维空间中实现了超越光学衍射极限的单分子荧光成像。DH - PSF设计具有高且均匀的费舍尔信息，在图像平面中有两个主要瓣，其角取向随发射体的轴向（z）位置旋转。通过找到两个DH - PSF瓣的中心，厚聚合物样品中的单个荧光分子在单次500毫秒采集过程中，能在大景深（2微米）范围内以10至20纳米的精度进行定位。通过使用光激活荧光团，利用DH - PSF显微镜对稀疏子集进行重复成像，可实现对高浓度分子在所有三维空间中的超分辨率成像。光学PSF设计与光激活荧光团的数字后处理相结合，为提高超越瑞利衍射极限的三维成像分辨率开辟了道路。
