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Multiphoton Imaging to Identify Grana, Stroma Thylakoid, and Starch Inside an Intact Leaf

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Multiphoton Imaging to Identify Grana, Stroma Thylakoid, and Starch Inside an Intact Leaf

Mei-Yu Chen et al. BMC Plant Biol.

Abstract

Background: 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.

Result: 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.

Conclusion: 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.

Figures

Figure 1
Figure 1
Sub-organelles inside a chloroplast. Grana and stroma thylakoid contains fluorescent chlorophyll. Both starch granules and grana exhibit stacking structures, allowing SHG conversion.
Figure 2
Figure 2
Transition diagrams of 2PF and SHG. (a) Two-photon excitation occurs through the absorption of two lower-energy photons via short-lived intermediate states. After excitation, the fluorophore first relaxes to the lowest energy level of the excited electronic states via fast vibrational processes, and then relaxes back to the ground state, emitting a spontaneous fluorescence photon. (b) In the process of SHG, two photons are annihilated and a single photon with doubled energy (half the wavelength) is generated. No real-state transition is required in SHG.
Figure 3
Figure 3
Multiphoton laser scanning microscopic images of chloroplasts inside an intact leaf. The backward 2PF and forward SHG signals are presented as red and green colors, respectively. We choose three different regions to illustrate the distribution and meaning of colors. For each region, a line profile corresponds to the white dashed line is shown to the right. (arrow: grana; arrowhead: starch; dotted arrow: stroma thylakoid) (a) The chloroplast is filled with 2PF (red) overlapped with discrete SHG (green) spots. In the combined image, the red part corresponds to stroma thylakoid, and the yellow (=green + red) part corresponds to grana. (b) The distribution of 2PF is complementary to SHG. In the combined image, the red part still corresponds to stroma thylakoid, and the green part shows the location of starch granules. (c) In the combined image, yellow, green, and red correspond to grana, starch, and stroma thylakoid, respectively.
Figure 4
Figure 4
Multiphoton imaging and fluorescence lifetime imaging of 2PF and SHG. The intensity profile and lifetime profile corresponding to the dashed line in the highlighted area are shown in the right-hand side. (a) Merged images of SHG and 2PF signals, similar to those in Figure 3 (b) and (c)In-situ measured 2PF and SHG lifetime imaging, respectively. The color bar shows the corresponding lifetime.
Figure 5
Figure 5
Schematic diagram of the experiment set-up. For intensity measurement, both backward and forward channels are used to detect 2PF and SHG signals, respectively. For lifetime measurement, a photon-counting PMT is placed in the forward direction, and connected to a TCSPC system. HWP: half-wave plate, PBS: polarization beam splitter, L: lens, BA6: BA610IF, FG: FGS600, BA5: BA565IF, F: FF520, FBG: FBG39. M1, M2: mirrors.

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