共路并行荧光辐射差分超分辨显微成像  

Common-Path Parallel Fluorescence Emission Difference Super-Resolution Microscopy

作　　者：张智敏 黄宇然 刘少聪[1] 匡翠方[1,2,3] 曹良才 刘勇[5] 韩于冰[1] 郝翔 刘旭[1] Zhang Zhimin;Huang Yuran;Liu Shaocong;Kuang Cuifang;Cao Liangcai;Liu Yong;Han Yubing;Hao Xiang;Liu Xu(State Key Laboratory of Modern Optical Instrumentation,College of Optical Science and Engineering,Zhejiang University,Hangzhou,Zhejiang 310027,China;Ningbo Research Institute,Zhejiang University,Ningbo,Zhejiang 315100,China;Collaborative Innovation Center of Extreme Optics,Shanxi University,Taiyuan,Shanxi 030006,China;State Key Laboratory of Precision Measurement Technology and Instrument,Department of Precision Instruments,Tsinghua University,Beijing 100084;Shanghai University of Electric Power,Shanghai 200090)

机构地区：[1]浙江大学光电科学与工程学院,现代光学仪器国家重点实验室,浙江杭州310027 [2]浙江大学宁波研究院,浙江宁波315100 [3]山西大学极端光学协同创新中心,山西太原030006 [4]清华大学精密测试技术及仪器国家重点实验室,北京100084 [5]上海电力学院,上海200090

出　　处：《中国激光》2021年第16期125-131,共7页Chinese Journal of Lasers

基　　金：国家自然科学基金(61827825,61735017);浙江省自然科学基金重大项目(LD21F050002);浙江省重点研究计划(2020C01116)。

摘　　要：本文提出了一种共路并行荧光辐射差分超分辨显微成像方法,利用单个空间光调制器(SLM)同时产生两个相位灰度图,对入射激光的水平偏振分量和垂直偏振分量同时进行调制,使得最终的会聚光场由错开的高斯实心光斑和空心光斑组成。在样品面上,利用错开的实心光斑和空心光斑同时对样品进行扫描,与此同时在探测端利用两个探测器同时收集错开光斑所激发的荧光信号,采用荧光辐射差分(FED)方法对采集的图像进行处理,就可以实现对样品的快速超分辨成像。与传统的并行荧光辐射差分超分辨显微术相比,本方法在保留了将成像速度提高一倍的优势的同时克服了非共路并行系统中不同器件引入的噪声、漂移对图像质量的影响,并简化了光路。实验结果表明所提共路并行荧光辐射差分超分辨显微成像方法具有良好的超衍射极限成像能力。Objective Fluorescence emission difference(FED)microscopy is a versatile super-resolution microscopy flexible for all types of fluorescent dyes.However,the limited imaging speed is the main drawback that hinders the application of FED,owing to the double imaging process of positive confocal image using a solid spot scan and negative confocal image using a negative spot scan.Parallel fluorescence microscopy can overcome this limitation owing to its ability to simultaneously capture positive and negative confocal images.However,the complexity of this method's system will increase instability and difficulties in daily maintenance,which also considerably restricts the popularization of the method.In this study,a new imaging method using a spatial light modulator(SLM)named common-path parallel FED(cpFED)microscopy was proposed.Compared with the traditional parallel FED microscopy,the proposed method that uses SLM and common path modulation maintains the advantage of doubling the imaging speed while overcoming the impact of instabilities introduced by different devices in noncommon-path parallel systems and simplifies the light path.Methods A property of SLM is that only linear polarized light can be modulated in a fixed direction,which is the major property of common-path parallel FED microscopy.Using the polarization rotation realized by passing forth and back through a quarter-wave plate,SLM can modulate the s and p polarization components of the emitted light(Fig.1).Using two-phase grayscale patterns,vortex and tilt-grating modulation patterns,to simultaneously modulate the horizontal and vertical polarization components of the incident laser beam on a single SLM,a staggering Gaussian solid spot and a hollow spot are generated to form the final convergent light field at the focus plane.The solid and hollow spots scan the sample simultaneously,and the fluorescence signal excited by the two spots is measured using two detectors,which will introduce a fixed transverse displacement between two images.Translating the po

关 键 词：显微 荧光显微镜 快速超分辨成像 荧光辐射差分技术 共路并行探测 

分 类 号：O436[机械工程—光学工程]