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。
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.
[1] White Amp J G, Amos W B. Confocal microscopy comes of age[J]. Nature, 1987, 328(6126):183-184. [2] White J G, Amos W B, Fordham M. An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy[J]. Journal of Cell Biology, 1987, 105(1):41-48. [3] Turillazzi E, Karch S B, Neri M, et al. Confocal laser scanning microscopy:Using new technology to answer old questions in forensic investigations[J]. Deutsche Zeitschrift Für Die Gesamte Gerichtliche Medizin, 2008, 122(2):173-177. [4] Wade M H. Fluorescence quantifi cati on in living cells[J]. Biomed Tech, 1992, 87:43-48. [5] 周涛, 杨怡, 张德添, 等. 激光扫描共聚焦显微镜及其在生物医学中的应用[J]. 军事医学科学院院刊, 2002, 1:69-73.ZHOU Tao, YANG Yi, ZHANG Detian, et al. Laser scanning confocal microscope and its applications in biomedicine[J]. Bulletin of The Academy of Military Medical Sciences, 2002, 1:69-73.(in Chinese) [6] Hong R. Observation of rice embryo sac development with confocal laser scanning microscopy[J]. Acta Botanica Sinica, 1998, 40(9):786-789. [7] Wang D, Wang X, Zhang W. Revealing the F-actin networks in interphase nuclei of garlic clove cells by confocal fluorescence microscopy[J]. Acta Botanica Sinica, 2000, 42(11):1167-1171. [8] Lamprecht A, Schäfer U F, Lehr C M. Characterization of microcapsules by confocal laser scanning microscopy:Structure, capsule wall composition and encapsulation rate[J]. European Journal of Pharmaceutics & Biopharmaceutics, 2000, 49(1):1-9. [9] Kubinova L, Janacek J, Karen P, et al.Confocal stereology and image analysis:Methods for estimating geometrical characteristics of cells and tissues from three-dimensional confocal images[J].Physiological Research, 2004, 53(Suppl 1):47-55. [10] 邱丽荣, 李佳, 赵维谦, 等. 激光共焦透镜曲率半径测量系统[J]. 光学精密工程, 2013, 21(2):246-252.QIU Lirong, LI Jia, ZHAO Weiqian, et al. Laser confocal measurement system for curvature radii of lenses[J].Optics and Precision Engineering. 2013, 21(2):246-252.(in Chinese) [11] 郭俊杰, 邱丽荣, 王允, 等. 用于惯性约束聚变靶丸测量的激光差动共焦传感器[J]. 光学精密工程, 2013, 21(3):644-651.GUO Junjie, QIU Lirong, WANG Yun, et al. Laser differential cofocal sensor for ICF capsule measurement[J]. Optics and Precision Engineering, 2013, 21(2):246-252.(in Chinese) [12] Leister D. Chloroplast research in the genomic age[J]. Trends in Genetics Tig, 2003, 19(1):47-56. [13] Kitada M, Ohsaki Y, Matsuda Y, et al. Photodynamic diagnoses of malignant pleural diseases using the autofluorescence imaging system[J]. Annals of Thoracic & Cardiovascular Surgery Official Journal of the Association of Thoracic & Cardiovascular Surgeons of Asia, 2014, 20(5):378-382. [14] Nichols A J, Evans C L. Video-rate scanning confocal microscopy and microendoscopy[J]. J Vis Exp, 2011(56), e3252. [15] Hanrahan O, Harris J, Egan C. Advanced microscopy:Laser scanning confocal microscopy[J]. Methods in Molecular Biology, 2011, 784(S 1):169-80. [16] Collings D A. Optimisation approaches for concurrent transmitted light imaging during confocal microscopy[J]. Plant Methods, 2015, 11(1):1-11. [17] 吴伟全, 李元歌, 王思捷, 等. CO<sub>2</sub>及温度对显微活细胞培养系统动态观察活细胞Ca<sup>2+</sup>变化的影响[J]. 实验与检验医学, 2012, 30(6):541-545.WU Weiquan, LI Yuange, WANG Sijie, et al.Effects of CO<sub>2</sub> and temperature modulation on dynamic change of Ca<sup>2+</sup> of living cells in a microscopic cell culture system[J]. Experimental and Laboratory Medicine, 2012, 30(6):541-545.(in Chinese) [18] 王秀玲, 彭清才. 叶绿体荧光观察方法的探讨[J]. 实验室科学, 2010, 13(4):76-78.WANG Xiuling, PENG Qingcai. Discussion on the observation of chloroplasts fluorescence[J]. Laboratory Science, 2010, 13(4):76-78.(in Chinese) [19] 刘向东. 利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化[J]. 激光生物学报, 2007, 16(2):173-178.LIU Xiangdong. A study on the developmental morphology of plant cell by laser scanning confocal Mi croscope[J]. Acta Laser Biology Sinica, 2007, 16(2):173-178.(in Chinese) [20] 崔永兰, 王鹏程, 杨仲南, 等. 拟南芥未知功能基因At3g61870编码蛋白的叶绿体定位研究[J]. 西北植物学报, 2008, 28(4):662-666.CUI Yonglan, WANG Pengcheng, YANG Zhongnan, et al[J]. Chloroplast localization of the expressed protein encoded by At3g61870 in arabidopsis thaliana[J]. Botanica Boreali-Occidentalia Sinica, 2008, 28(4):662-666.(in Chinese)
