中国激光, 2013, 40 (11): 1110001, 网络出版: 2013-10-20
基于宽场随机荧光漂白的超分辨显微方法
Superresolution Microscopy Imaging Based on Wide-Field Stochastic Fluorescent Bleaching
显微 超分辨 随机荧光漂白 单分子定位 microscopy superresolution stochastic fluorescent bleaching localization of single molecule
摘要
对基于宽场随机荧光漂白的超分辨显微方法进行了研究。通过对获取的荧光漂白图像进行差分处理,获得大量稀疏的荧光分子图像,相应的荧光分子被一一区分并通过点扩散函数(PSF)数字化的方法计算出中心位置,从而重构超分辨显微图像。并对标记Alex 488普通荧光染料的人胚肾细胞进行了成像,和普通荧光显微镜相比,分辨率提升了约40%,表明该方法适用于常规荧光染料,并能突破衍射极限达到超分辨效果。该方法克服了常用的基于单分子精确定位技术需要对荧光分子进行反复地激活猝灭,并难以适用于普通荧光染料的缺点。
Abstract
A superresolution imaging technique based on wide-field stochastic fluorescent bleaching is studied. A stream of fluorescence images is simply acquired, and single fluorophore is detected by subtracting each frame from the previous one. After image subtractions, a superresolution image is got by fitting images of single fluorescent molecules with a point dispersion function method to localize them. Then this method is used to image the human embryonic kidney cells with the conventional fluorescent probe Alex 488. Compared with the conventional fluorescence microscope, the resolution increases about 40%, showing that it works with the sample with conventional fluorescent probes and can break the diffraction limit. Also it overcomes the weakness of conventional techniques based on the precise localization of single molecules, which needs to activate and quench the fluorescent molecules repeatedly, and can not be applied to conventional fluorescent samples.
蔡欢庆, 匡翠方, 王轶凡, 葛剑虹, 刘旭. 基于宽场随机荧光漂白的超分辨显微方法[J]. 中国激光, 2013, 40(11): 1110001. Cai Huanqing, Kuang Cuifang, Wang Yifan, Ge Jianhong, Liu Xu. Superresolution Microscopy Imaging Based on Wide-Field Stochastic Fluorescent Bleaching[J]. Chinese Journal of Lasers, 2013, 40(11): 1110001.