Department of Physics, National Taiwan University, Taipei, Taiwan.
BMC Plant Biol. 2014 Jun 27;14:175. doi: 10.1186/1471-2229-14-175.
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.
类囊体基粒和淀粉粒分别是植物光合作用和能量储存的主要功能结构。它们都具有高度有序的分子结构，在叶绿体中呈现为微米级颗粒。为了区分基粒和淀粉粒，我们使用多光子显微镜，同时获取双光子荧光（2PF）和二次谐波产生（SHG）信号。SHG 对结晶结构敏感，而 2PF 则选择性地揭示叶绿素的分布。
观察到三种具有不同对比度的不同微观结构，即“SHG 占主导”、“2PF 占主导”和“SHG 与 2PF 共定位”。已知淀粉粒和基粒都由于其高度结晶的结构而发出 SHG，并且淀粉粒不发出自发荧光，因此“SHG 占主导”的对比度应该对应于淀粉粒。“SHG 与 2PF 共定位”的对比度被分配给基粒，基粒具有结晶结构和自体荧光叶绿素。“2PF 占主导”的对比度应该对应于基质类囊体，它是一种具有叶绿素的非堆积膜结构。荧光寿命测量进一步支持了对比度的分配。
我们已经证明了一种简单、非侵入性的方法，可以识别完整叶片中基粒和淀粉粒的分布。通过合并 2PF 和 SHG 图像，可以直观地区分基粒、淀粉粒和基质类囊体。这种方法可以扩展到观察活植物中 3D 基粒分布及其动态。
