Multiphoton imaging to identify grana, stroma thylakoid, and starch inside an intact leaf
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 19 July 2013
Accepted: 18 June 2014
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.
KeywordsGrana Starch Two-photon fluorescence Second harmonic generation
The three-dimensional arrangement of grana and starch granules in a chloroplast has been observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) [1–6]. As expected, TEM and SEM provide very high spatial resolution down to nanometer scale. Nevertheless, both TEM and SEM need sophisticated specimen preparation. TEM specimens cannot be thicker than hundreds of nanometers, so it is not practical for TEM to perform whole-leaf observation. For SEM, since the chamber is at high vacuum, plant specimen is normally required to be fully dehydrated by chemical fixation, which may cause molecular denaturation and structure artifact. The freeze-fracture approach for SEM sample processing might authentically display the fine structures of the chloroplast, but it cannot be used in live cell applications.
Optical microscopy, on the other hand, exhibits much lower spatial resolution (about half of visible wavelength), but it can be used to view cells that are living and functioning. Conventional compound microscopes are not capable of producing a clear three-dimensional view in thick biological tissues. By combining a confocal pinhole with laser scanning microscopy, confocal microscopy provides excellent optical sectioning capability. In the late 1980s, confocal microscopy was adopted to study the inner structures of chloroplasts in living and intact plants based on the autofluorescence from chlorophyll [7, 8]. However, since both grana and stroma thylakoid contain chlorophyll, it is difficult to distinguish them simply by fluorescence intensity, unless using sophisticated spectral imaging microscope [9, 10]. Since grana are well-packed thylakoids, a contrast agent that is sensitive to crystallization will be useful to distinguish grana and stroma thylakoids. One possible solution is second harmonic generation, which is a nonlinear optical process that is allowed only in non-centrosymmetric structures .
In the case of SHG, laser light is focused on a sample to generate frequency-doubled light. During SHG process, two incident photons are annihilated and a new photon is generated. Due to energy conservation, the energy, and thus frequency, of the new photon is twice of that of the annihilated photons, as shown in Figure 2(b). One advantage of SHG is that only virtual state transition is involved, thus no photo-damage and photon-bleaching are expected, since no energy is deposited during this transition . On the other hand, SHG also exhibits a square dependence on incident intensity, so SHG imaging provides excellent optical sectioning capability, similar to 2PF imaging. In principle, SHG is sensitive to non-centrosymmetric crystallized media, such as an interface between two centrosymmetric media , and crystallized structures, such as well-packed biological structures, including collagen and myosin in animal, as well as starch and grana in plant [11, 16–27]. Inside a chloroplast, both starch and grana exhibit SHG, so in a previous work , the authors identified SHG of grana by keeping the plants in the dark for approximately 3 weeks to devoid starch content in the plant. However, this process might severely affect plant physiology, and the method prevents study of photosynthesis under normal illumination condition.
The aim of this paper is to demonstrate that nonlinear optical microscopy is a noninvasive method to identify the distribution of grana, stroma thylakoid, and starch granules within a live intact leaf. No complicated sample preparation or darkroom process is required. Through simultaneous acquisition of 2PF and SHG signals, these cellular organelles can be visually identified. This novel approach can be used in the field of botanical evolutionism, and provides a dynamic imaging observation in the growth of the plants.
In Figure 3(b), the distribution of SHG and 2PF are complementary to each other. As can be seen in the 2PF image, there are cavities within each chloroplast. In the SHG image, there are multiple bright spots showing the location of crystallized structures. In the combined image, as well as in the line profile, we see that the 2PF cavities are filled with bright SHG spots. Again, for those regions in red color, they represent stroma thylakoid. On the other hand, for those regions with SHG but no 2PF, they exhibit green color and should correspond to crystallized structures without autofluorescence in chloroplast, i.e. starch [11, 23].In Figure 3(c), some SHG spots are collocated with 2PF, while others are not. Based on previous discussion, the green spots (SHG dominates) indicate the location of starch granules; the yellow color (SHG collocated with 2PF) provides the distribution of grana, while the red color (2PF dominates) corresponds to stroma thylakoid. It is interesting to notice that typically chloroplasts are in a round to elliptical shape, but the 2PF image here shows an irregular outline. It has been known from the electron microscopy studies that stroma thylakoid is not evenly distributed inside chloroplast, so the fact that 2PF is weak in some area reflects the low concentration of stroma thylakoid.
In addition, an axial image series (see Additional file 1) is given in the movie, demonstrating excellent optical sectioning capability of nonlinear optical microscopy in plant leaves. Based on the discussion above, the distribution of grana, starch, and stroma thylakoid can be identified by color of yellow, green, and red, respectively.
The table gives a summary of 2PF and SHG signals correspond to three different kinds of structures, including grana, starch and stroma thylakoid
One possible ambiguity lies in the interpretation of overlapped SHG and 2PF. Our current explanation is that the overlapping reflects the coexistence of chlorophyll autofluorescence and stacked structures, i.e. grana. However, it is possible that the structure we assigned to be a granum might be a starch granule surrounded by stroma thylakoid. Here we describe the reason why the latter interpretation is more unlikely.Let's examine Figure 3(a) more closely. Based on our experimental condition (objective and wavelength), the lateral and axial resolutions are better than 0.5 and 1 μm, respectively. The size of a granum and a starch granule in our sample should be about 1 μm, since we used a shade plant (see Methods). The sizes of SHG spots are larger than 1 μm in Figure 3(a). If these SHG spots are large starch granules (i.e. larger than 1 μm) surrounded by stroma thylakoid, the 2PF signals should significantly drop at the center of the SHG spots. Nevertheless, we did not observe this in Figure 3(a). On the other hand, it might be a very tiny starch granule that generate SHG but overlapped with 2PF from surrounding stroma thylakoid. However, it is also unlikely because the SHG spots we observed in Figure 3(a) are more than 1 μm in width, which is larger than the resolution limit of our multiphoton system. It is evident that we can distinguish the orientation of the two selected SHG microparticles in Figure 3(a), and it serves as a proof that the optical resolution is better than the size of the microparticles. In fact, from the intensity profiles of Figure 3(a), the fluorescence signals actually increase along with SHG in the particles. So we conclude that the selected SHG particles in Figure 3(a) should correspond to grana, not starch granules.On the other hand, for the structure that we assigned to be a starch granule, could it be a granum? The first thing to note is that fluorescence intensity reflects local density of chlorophyll, and the volume density of chlorophyll is higher in grana compared to surrounding stroma thylakoid. So the fluorescence intensity of grana should be no less than that of stroma thylakoid. As shown in Figure 3(b), if the SHG spots correspond to grana, the 2PF intensity at the location of SHG spots should be stronger or at least equal to the surrounding 2PF intensity. However, what we found in Figure 3(b) are particles with strong SHG and reduced 2PF signals. So they should be starch granules, not grana. The residual weak 2PF signals at the SHG spot might come from the stroma thylakoid adjacent to the starch granule in the axial direction.From Figure 3(c), in the region of grana, both 2PF and SHG increase compared to the surrounding; while in the region of starch, a strong SHG peak is observed with significantly reduced 2PF. This gives strong support that our technique can indeed separate starch and grana.
We have demonstrated a noninvasive method to identify the distribution of grana and starch inside a live mesophyll cell without any special specimen labeling or handling. The method is based on the combination of SHG and 2PF contrast in a multiphoton microscope. There are two types of SHG structures inside a chloroplast of a plant cell. One is collocated with strong 2PF, and the other is complementary to 2PF. The former correspond to grana while the latter correspond to starch. For those regions with only 2PF but no SHG, they represent the distribution of stroma thylakoid. By merging 2PF and SHG images, the grana, starch granules, and stroma thylakoid can be visually distinguished by different colors of yellow (green + red), green, and red, respectively. The structure identification is further proved by fluorescence lifetime measurements. The nonlinear nature of the multiphoton process provides useful intrinsic optical sectioning capability and is less likely to cause damage in live sample, enabling observation of organelle dynamics during plant growth. Our technique will be useful to study granal structural variation among different plant specie , and can be used in the field of botanical evolutionism.
The leaf we used here was detached from a fresh ferns, Macrothelypteris torresiana (Gaud.) Ching, which belongs to shaded plants with large grana [33–37]. The leaf was mounted in water between a coverslip and a glass slide. The edges of the coverslip were sealed by nail varnish. The glass slide was placed on the microscope stage for observation.
For fluorescence lifetime measurement, the excitation and scanning systems are the same as above, but the detection part becomes a photon-counting PMT (PMC-100-1, Becker & Hickl, Germany) in the forward direction, equipped with a time-correlated single photon counting system (TCSPC-150, Becker and Hickl, Germany). A high-speed photodetector synchronize the laser repetition rate to the photon counting system. During lifetime measurement, corresponding filters are placed in front of the photon counting PMT to allow either 2PF or SHG detection without cross talk.
This work is supported by the Ministry of Science and Technology of ROC under contract No. NSC-102-2112-M-002 -018 -MY3 and NSC- 101-2923-M-002-001-MY3.
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