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清华大学学报(自然科学版)
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利用激光扫描共聚焦显微镜研究叶绿体自发荧光
冯倩倩1,2, 王文娟1,3, 李海冬1,2, 潘勋1,2
1. 清华大学 生命科学学院, 北京 100084;
2. 清华大学 生物医学测试中心, 北京 100084;
3. 清华大学 蛋白质研究技术中心, 北京 100084
Autofluorescence of chloroplasts measured by a laser scanning confocal microscope
FENG Qianqian1,2, WANG Wenjuan1,3, LI Haidong1,2, PAN Xun1,2
1. School of Life Sciences, Tsinghua University, Beijing 100084, China;
2. Center of Biomedical Analysis, Tsinghua University, Beijing 100084, China;
3. Technology Center for Protein Sciences, Tsinghua University, Beijing 100084, China
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摘要 该文利用激光扫描共聚焦显微镜研究烟草叶片叶绿体自发荧光,为叶绿体研究提供理论和实验依据。首先,利用不同波长激发光的光谱扫描功能分别激发烟草叶片叶绿体,得到叶绿体自发荧光光谱;其次,利用不同激发光激发烟草叶片叶绿体,获取叶绿体自发荧光图像,并对荧光强度进行定量分析比较;最后,利用488 nm激发光漂白烟草叶片叶绿体,观察自发荧光的稳定性。实验结果显示: 488 nm激光对叶绿体的激发效率最高,561 nm激光的激发效率最低;发射光谱集中在大于637 nm的波段,峰值在681 nm处;且叶绿体自发荧光非常稳定。激光扫描共聚焦显微镜能够准确研究叶绿体自发荧光,对研究植物组织成像提供重要帮助,也对叶绿体基因工程研究有深远影响。
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冯倩倩
王文娟
李海冬
潘勋
关键词 激光扫描共聚焦显微镜叶绿体自发荧光光谱扫描    
Abstract:The autofluorescence of nicotiana benthamiana chloroplasts was studied using a laser scanning confocal microscope. The chloroplast autofluorescence was obtained by spectral scanning with various excitation wavelengths. Then, chloroplast autofluorescence images were obtained using laser scanning confocal microscopy with various excitation wavelengths with intensity quantification. Finally, nicotiana benthamiana chloroplasts were illuminated with 488 nm light to test the autofluorescence stability. 488 nm light has the highest excitation efficiency for chloroplast autofluorescence, while 561 nm light has the lowest excitation efficiency. The autofluorescence chloroplast emission spectrum starts from 637 nm and peaks at 681 nm. The measurements show that the chloroplast autofluorescence is very stable and that laser scanning confocal microscopy can accurately measure chloroplasts autofluorescence. This work will facilitate improved plant tissue imaging chloroplast genetic engineering research.
Key wordslaser scanning confocal microscopy    chloroplast    autofluorescence    spectral imaging
收稿日期: 2016-11-14      出版日期: 2017-06-15
ZTFLH:  TH742  
通讯作者: 潘勋,研究员,E-mail:panx@biomed.tsinghua.edu.cn     E-mail: panx@biomed.tsinghua.edu.cn
引用本文:   
冯倩倩, 王文娟, 李海冬, 潘勋. 利用激光扫描共聚焦显微镜研究叶绿体自发荧光[J]. 清华大学学报(自然科学版), 10.16511/j.cnki.qhdxxb.2017.26.034.
FENG Qianqian, WANG Wenjuan, LI Haidong, PAN Xun. Autofluorescence of chloroplasts measured by a laser scanning confocal microscope. Journal of Tsinghua University(Science and Technology), 2017, 57(6): 651-654,660.
链接本文:  
http://jst.tsinghuajournals.com/CN/10.16511/j.cnki.qhdxxb.2017.26.034  或          http://jst.tsinghuajournals.com/CN/Y2017/V57/I6/651
  表1 激光扫描共聚焦显微镜参数设定
  图1 不同波长激光下烟草叶片叶绿体自发荧光的荧光光谱
  图2 烟草叶片叶绿体自发荧光图像,激光功率0.05mW,接收波长637~758nm
  图3 不同激光波长下烟草叶片叶绿体自发荧光的平均荧光强度
  图4 烟草叶片叶绿体自发荧光强度变化
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