摘要
目的:探讨激光共聚焦显微镜不同参数对活细胞荧光漂白的影响。方法:采用不同的激光共聚焦显微镜拍摄参数对钙黄绿素标记的骨肉瘤细胞进行时间序列成像,分析随拍摄帧数增多荧光强度的变化。结果:激光功率增大、激光驻留时间延长都会增加荧光漂白,当保持激光驻留时间与激光强度的乘积(即照射在样品上的总能量)不变时,驻留时间最长的组荧光漂白率最高(P<0.05),驻留时间最短的组荧光漂白率最低(P<0.05);NA值相似的情况下,20倍物镜结合ZOOM=2的模式比直接用40倍物镜拍摄荧光漂白率更高;细胞在氧含量3%的培养条件中预处理1 h,与常规培养条件比,在拍摄早期加剧光漂白,在拍摄后期延缓光漂白。结论:对活细胞进行三维重建或时间序列成像时,建议先通过预试验找到光漂白率最小且同时满足分辨率需求的拍摄参数组合,可以考虑高激光功率、短驻留时间的组合;非必须情况,尽量避免使用ZOOM模式;如何利用低氧条件缓解对活细胞的光漂白尚需进一步探索。
Objective:To investigate the effects of different parameters of laser confocal microscopy on photo-bleaching of living cells.Methods:Time lapse images of osteosarcoma cells with calcein-AM labeled were performed using different laser scanning parameters,and the changes in fluorescence intensity while the increase of scanning frames were analyzed.Results:Photo-bleaching rate of the group with the longest frame dwell time was the highest(P<0.05),and that of the group with the shortest dwell time was the lowest(P<0.05);When NAs were similar,the bleaching rate of 20×objective combined with zoom=2 mode was higher than that of using 40×objective directly;Photo bleaching of cells pretreated for 1 hour in 3%oxygen content,compared with in the conventional culture conditions,the effect was intensified in the early stage of scanning and delayed in the later stage.Conclusion:For 3 D reconstruction or time series imaging of living cells,it is necessary to find out the optimal parameter combination,which with the minimum photo-bleaching rate and meet the resolution requirements through pretest.The combination of high laser power and short dwell time should be considered;Zoom mode should be avoided as far as possible if it is not necessary;How to use hypoxia to alleviate photo-bleaching effects needs further exploration.
作者
杨桢
陵廷贤
张杰
YANG Zhen;LING Ting-xian;ZHANG Jie(The Seventh Affiliated Hospital,Sun Yat-Sen University,Shenzhen Guangdong 518107;Core Facility of West China Hospital,Sichuan University,Chengdu Sichuan 610041;Department of Orthopedics,West China Hospital,Sichuan University,Chengdu Sichuan 610041,China)
出处
《电子显微学报》
CAS
CSCD
北大核心
2021年第6期704-710,共7页
Journal of Chinese Electron Microscopy Society
基金
四川省科技计划应用基础研究(No.2020YJ0063)。
