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
Fluorescence recovery after photobleaching(FRAP)has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997.Fluorescence recovery of cyanobacteria during FRAP was conventionally inter...Fluorescence recovery after photobleaching(FRAP)has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997.Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome(PBS)diffusion on the surface of the thylakoid membrane.The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion.However,in red algae,another PBS-containing group,the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS,which raises questions concerning the role of FRAP in red algal PBS.Therefore,it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria.In the present study,four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics.Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains,in which the contribution of PBS diffusion to the fluorescence recovery was not possible.Moreover,the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery.These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.展开更多
药物分子从药物载体中的释放行为与载体的结构有密切关系.本实验中采用丙烯酰胺和丙烯酸等材料,运用水相沉淀的方法,制备了4种不同单体配比的聚丙烯酰胺-丙烯酸(P(Am-co-Ac))共聚物水凝胶.运用红外分析方法对P(Am-co-Ac)组成进行表征....药物分子从药物载体中的释放行为与载体的结构有密切关系.本实验中采用丙烯酰胺和丙烯酸等材料,运用水相沉淀的方法,制备了4种不同单体配比的聚丙烯酰胺-丙烯酸(P(Am-co-Ac))共聚物水凝胶.运用红外分析方法对P(Am-co-Ac)组成进行表征.使用荧光漂白恢复法(FRAP,fluorescence recovery after photobleaching)观察荧光素FITC标记的牛血清白蛋白(BSA)在聚丙烯酰胺-丙烯酸中的扩散行为,并以激光共聚焦显微镜进行实时成像.实验表明,FITC-BSA在不同单体配比的共聚物中的扩散系数是不同的.通过调节聚合物中单体的配比能够达到控制蛋白释放速率的作用,从而为调控蛋白和多肽类药物的缓控释放提供了可能性.展开更多
目的:探讨重组腺病毒介导KDR-CDglyTK系统杀伤胃癌SCG7901细胞时细胞缝隙连接与旁观者效应的关系.方法:感染复数(multiplicity of infection,MOI)=100时,将携带融合基因重组腺病毒体外感染SCG7901、HeLa细胞,检测细胞的感染效率以及转...目的:探讨重组腺病毒介导KDR-CDglyTK系统杀伤胃癌SCG7901细胞时细胞缝隙连接与旁观者效应的关系.方法:感染复数(multiplicity of infection,MOI)=100时,将携带融合基因重组腺病毒体外感染SCG7901、HeLa细胞,检测细胞的感染效率以及转基因肿瘤细胞在mRNA水平上融合基因的表达;用荧光漂白恢复技术(fluorescence recovery after photobleaching,FRAP)检测SCG7901、HeLa细胞以及芹黄素作用上述两种细胞的细胞间通讯功能.将已转染细胞和未转染细胞分别按10%∶90%和5%∶95%两种比例混合,用含或不含芹黄素的培养基,培养24 h后,在不同组中分别加入GCV、5-FC两者的混合液,72 h后用MTT法检测细胞存活率.结果:受感染的SCG7901和HeLa细胞中均有绿色荧光蛋白的表达.图像中观察到在芹黄素作用下SCG7901细胞淬灭后荧光强度明显降低,随后随着时间的推移,被淬灭细胞的荧光强度逐渐恢复,在相同时间内其恢复强度较未应用上调剂的SCG7901细胞要强;而在芹黄素作用下的HeLa细胞被淬灭后荧光强度明显降低,随时间的推移荧光强度无明显恢复,在上调剂芹黄素作用下的SCG7901与HeLa细胞间的荧光恢复率有差异.同时,观察到双自杀基因系统在转基因细胞比例分别占5%、10%时,SCG7901细胞在空白组、前药组、芹黄素组、前药+芹黄素组间细胞生存率有差异(F=144.42,P=0.000)、(F=407.83,P=0.000),而HeLa细胞在各组间的细胞生存率无统计学意义(F=0.386,P=0.765)、(F=0.895,P=0.472).结论:FRAP技术证实SCG7901细胞间通讯功能与缝隙连接有一定关系,而HeLa细胞不存在细胞间通讯功能.双自杀基因系统治疗SCG7901细胞时存在旁观者效应而且上调剂可以扩大其旁杀效应;但HeLa细胞不存在旁观者效应.双自杀基因系统治疗胃癌细胞时旁观者效应与细胞间缝隙连接有关.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.
基金This work was supported by the National Natural Science Foundation of China(no.31900023)National Key R&D Program of China(no.2018YFC1406701)+4 种基金Program of Shandong Taishan Scholars(no.tspd20181203)Natural Science Foundation of Shandong(no.ZR2017LD013)AoShan Talents Cultivation Program(no.2017ASTCP-OS14)State Key Laboratory of Microbial Technology Open Projects Fund(no.M2019-07)Young Scholars Program of Shandong University(no.2017WLJH22).
文摘Fluorescence recovery after photobleaching(FRAP)has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997.Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome(PBS)diffusion on the surface of the thylakoid membrane.The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion.However,in red algae,another PBS-containing group,the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS,which raises questions concerning the role of FRAP in red algal PBS.Therefore,it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria.In the present study,four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics.Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains,in which the contribution of PBS diffusion to the fluorescence recovery was not possible.Moreover,the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery.These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.
文摘药物分子从药物载体中的释放行为与载体的结构有密切关系.本实验中采用丙烯酰胺和丙烯酸等材料,运用水相沉淀的方法,制备了4种不同单体配比的聚丙烯酰胺-丙烯酸(P(Am-co-Ac))共聚物水凝胶.运用红外分析方法对P(Am-co-Ac)组成进行表征.使用荧光漂白恢复法(FRAP,fluorescence recovery after photobleaching)观察荧光素FITC标记的牛血清白蛋白(BSA)在聚丙烯酰胺-丙烯酸中的扩散行为,并以激光共聚焦显微镜进行实时成像.实验表明,FITC-BSA在不同单体配比的共聚物中的扩散系数是不同的.通过调节聚合物中单体的配比能够达到控制蛋白释放速率的作用,从而为调控蛋白和多肽类药物的缓控释放提供了可能性.
文摘目的:探讨重组腺病毒介导KDR-CDglyTK系统杀伤胃癌SCG7901细胞时细胞缝隙连接与旁观者效应的关系.方法:感染复数(multiplicity of infection,MOI)=100时,将携带融合基因重组腺病毒体外感染SCG7901、HeLa细胞,检测细胞的感染效率以及转基因肿瘤细胞在mRNA水平上融合基因的表达;用荧光漂白恢复技术(fluorescence recovery after photobleaching,FRAP)检测SCG7901、HeLa细胞以及芹黄素作用上述两种细胞的细胞间通讯功能.将已转染细胞和未转染细胞分别按10%∶90%和5%∶95%两种比例混合,用含或不含芹黄素的培养基,培养24 h后,在不同组中分别加入GCV、5-FC两者的混合液,72 h后用MTT法检测细胞存活率.结果:受感染的SCG7901和HeLa细胞中均有绿色荧光蛋白的表达.图像中观察到在芹黄素作用下SCG7901细胞淬灭后荧光强度明显降低,随后随着时间的推移,被淬灭细胞的荧光强度逐渐恢复,在相同时间内其恢复强度较未应用上调剂的SCG7901细胞要强;而在芹黄素作用下的HeLa细胞被淬灭后荧光强度明显降低,随时间的推移荧光强度无明显恢复,在上调剂芹黄素作用下的SCG7901与HeLa细胞间的荧光恢复率有差异.同时,观察到双自杀基因系统在转基因细胞比例分别占5%、10%时,SCG7901细胞在空白组、前药组、芹黄素组、前药+芹黄素组间细胞生存率有差异(F=144.42,P=0.000)、(F=407.83,P=0.000),而HeLa细胞在各组间的细胞生存率无统计学意义(F=0.386,P=0.765)、(F=0.895,P=0.472).结论:FRAP技术证实SCG7901细胞间通讯功能与缝隙连接有一定关系,而HeLa细胞不存在细胞间通讯功能.双自杀基因系统治疗SCG7901细胞时存在旁观者效应而且上调剂可以扩大其旁杀效应;但HeLa细胞不存在旁观者效应.双自杀基因系统治疗胃癌细胞时旁观者效应与细胞间缝隙连接有关.
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。