双通道结构光照明超分辨定量荧光共振能量转移成像系统  

Dual-channel structured illumination super-resolution quantitative fluorescence resonance energy transfer imaging

作　　者：罗泽伟 武戈 陈挚 邓驰楠 万蓉 杨涛 庄正飞 陈同生 Luo Ze-Wei;Wu Ge;Chen Zhi;Deng Chi-Nan;Wan Rong;Yang Tao;Zhuang Zheng-Fei;Chen Tong-Sheng(Key Laboratory of Laser Life Science,Ministry of Education,College of Biophotonics,South China Normal University,Guangzhou 510631,China;Guangdong Key Laboratory of Laser Life Science,College of Biophotonics,South China Normal University,Guangzhou 510631,China)

机构地区：[1]华南师范大学生物光子学研究院,教育部激光生命科学重点实验室,广州510631 [2]华南师范大学生物光子学研究院,广东省激光生命科学重点实验室,广州510631

出　　处：《物理学报》2023年第20期276-285,共10页Acta Physica Sinica

基　　金：国家自然科学基金(批准号:62135003);广东省重点领域研发计划(批准号:2022B0303040003)资助的课题.

摘　　要：基于结构光照明(structured illumination,SI)的超分辨荧光共振能量转移(super resolution fluorescence resonance energy transfer,SR-FRET)成像技术(SISR-FRET)可以通过解析活细胞内亚衍射区域的FRET信号来研究细胞器精细结构上的分子结构与功能.SISR-FRET成像中激发发射通道切换导致成像速度较慢,限制了SISR-FRET在快速成像中的应用.针对此问题,本文提出一种双通道结构光照明超分辨定量FRET成像系统和方法,通过在成像光路中加入FRET双通道成像和配准模块,实现了SISR-FRET激发发射通道的空分切换以及通道复用.结合通道亚像素配准校正的图像重建算法,双通道SISR-FRET可以在保持定量超分辨FRET分析的同时提升了3.5倍时间分辨率.利用搭建的多色SIM系统进行了活细胞表达靶向线粒体外膜FRET标准质粒的超分辨成像实验,验证了双通道SISR-FRET的时空分辨率增强和FRET定量分析的保真度.The Structured illumination(SI)-based super resolution fluorescence resonance energy transfer(SR-FRET)imaging technique,known as SISR-FRET,enables the investigation of molecular structures and functions in cellular organelles by resolving sub-diffraction FRET signals within living cells.The FRET microscopy offers unique advantages for quantitatively detecting dynamic interactions and spatial distribution of biomolecules within living cells.The spatial resolution of conventional FRET microscopy is limited by the diffraction limit,and it can only capture the average behavior of these events within the resolution limits of conventional fluorescence microscopy.The SISR-FRET performs sequential linear reconstruction of the three-channel SIM images followed by FRET quantitative analysis by using a common localization mask-based filtering approach.This two-step process ensures the fidelity of the reconstructed SR-FRET signals while effectively removing falsepositive FRET signals caused by SIM artifacts.However,the slow imaging speed resulting from the switching of excitation-emission channels in SISR-FRET imaging limits its application in fast imaging scenarios.To address this issue,this study proposes a dual-channel structured illumination super-resolution quantitative FRET imaging system and method.By incorporating an FRET dual-channel imaging and registration module into the imaging pathway,the spatial switching and channel multiplexing of the SISR-FRET excitation-emission channels are achieved.Combining the image reconstruction algorithm with channel sub-pixel registration correction,the dual-channel SISR-FRET technique enhances the temporal resolution by 3.5 times while preserving the quantitative super-resolution FRET analysis.Experimental results are obtained by using a multicolor SIM system to perform super-resolution imaging of living cells expressing mitochondria outer membrane FRET standard plasmids.These experiments validate the improved spatial and temporal resolution of dualchannel SISR-FRET and the fideli

关 键 词：荧光共振能量转移 结构光照明 超分辨 活细胞 

分 类 号：TP391.41[自动化与计算机技术—计算机应用技术] O439[自动化与计算机技术—计算机科学与技术]