Multiphoton imaging to identify grana, stroma thylakoid, and starch inside an intact leaf

BMC Plant Biology

Methodology Article

Kuan-Chieh Chen¹  Guan-Yu Zhuo¹  Mei-Yu Chen¹  Shi-Wei Chu²  Pei-Chun Wu³  Tsung-Yuan Hsieh³  Tzu-Ming Liu⁴ 

[1] Department of Physics, National Taiwan University, Taipei, Taiwan;Department of Physics, National Taiwan University, Taipei, Taiwan;Molecular Imaging Center, National Taiwan University, Taipei, Taiwan;Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan;Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan;Molecular Imaging Center, National Taiwan University, Taipei, Taiwan;

关键词: Grana;    Starch;    Two-photon fluorescence;    Second harmonic generation;   

DOI  :  10.1186/1471-2229-14-175

 received in 2013-07-19, accepted in 2014-06-18,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundGrana and starch are major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two-photon fluorescence (2PF) and second harmonic generation (SHG) signals. SHG is sensitive to crystallized structures while 2PF selectively reveals the distribution of chlorophyll.ResultThree distinct microstructures with different contrasts were observed, i.e. “SHG dominates”, “2PF dominates”, and “SHG collocated with 2PF”. It is known that starch and grana both emit SHG due to their highly crystallized structures, and no autofluorescence is emitted from starch, so the “SHG dominates” contrast should correspond to starch. The contrast of “SHG collocated with 2PF” is assigned to be grana, which exhibit crystallized structure with autofluorescent chlorophyll. The “2PF dominates” contrast should correspond to stroma thylakoid, which is a non-packed membrane structure with chrolophyll. The contrast assignment is further supported by fluorescence lifetime measurement.ConclusionWe have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. By merging the 2PF and SHG images, grana, starch and stroma thylakoid can be visually distinguished. This approach can be extended to the observation of 3D grana distribution and their dynamics in living plants.

【 授权许可】

Unknown   
© Chen et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.