Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of l...Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules,respectively.Lateral dif-fusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger dif-fusion coefficient determined by FRAP,but the sources of the inconsistency have not been investigated thoroughly.Here,we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes-Einstein equation in aqueous solution.The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis.Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.展开更多
激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 n...激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。展开更多
基金This work was supported by grants from the National Research Foundation(NRF)(NRF2019R1A2C2088973)funded by the Ministry of Educationthe Korea Evaluation Institute of Industrial Technology(KEIT)(20011377)funded by the Ministry of Trade,Industry&Energy,Republic of Korea.
文摘Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules,respectively.Lateral dif-fusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger dif-fusion coefficient determined by FRAP,but the sources of the inconsistency have not been investigated thoroughly.Here,we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes-Einstein equation in aqueous solution.The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis.Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.
文摘激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。
