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Open Access Methodology article

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

Mei-Yu Chen1, Guan-Yu Zhuo1, Kuan-Chieh Chen1, Pei-Chun Wu2, Tsung-Yuan Hsieh2, Tzu-Ming Liu23 and Shi-Wei Chu13*

Author Affiliations

1 Department of Physics, National Taiwan University, Taipei, Taiwan

2 Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan

3 Molecular Imaging Center, National Taiwan University, Taipei, Taiwan

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BMC Plant Biology 2014, 14:175  doi:10.1186/1471-2229-14-175

Published: 27 June 2014



Grana 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.


Three 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.


We 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.

Grana; Starch; Two-photon fluorescence; Second harmonic generation