Size-Dependent Optical Properties of Grana Inside Chloroplast of

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Size-Dependent Optical Properties of Grana Inside Chloroplast of Plant Cells Takayuki Uwada, Ling-Ting Huang, Ping-Yu Hee, Anwar Usman, and Hiroshi Masuhara J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10204 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 14, 2017

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The Journal of Physical Chemistry

Size-Dependent Optical Properties of Grana Inside Chloroplast of Plant Cells

Takayuki Uwada,†,§,* Ling-Ting Huang,† Ping-Yu Hee,† Anwar Usman,‡,* Hiroshi Masuhara†,*

†Department of Applied Chemistry, College of Science, National Chiao Tung University, 1001 Ta Hsueh Rd., Hsinchu 30010, Taiwan §Department of Chemistry, Faculty of Science, Josai University, Sakado 350-0295, Japan

‡Department of Chemistry, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong BE1410, Negara Brunei Darussalam

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ABSTRACT

Well-packed thylakoids known as grana are one of the major functional sites for photosynthesis in algae and plants. Their highly ordered structures can be considered as a few hundreds nanometer-sized particles having distinct scattering cross sections from other various macromolecular organizations inside plant cells. With this background we show that elastic light scattering imaging and microscopy is an important tool for investigating structure and organization of grana inside a single chloroplast in plant cells. We have demonstrated this noninvasive method to identify the distribution of grana in intact fresh leaf of robust and rapidly growing Egaria densa, which is also known as Anachris and among the most popular aquarium plants. The scattering efficiency spectra of their individual grana fairly resemble cooperative absorption spectra of porphyrins and carotenoids. We found that the electronic structure of the stacked thylakoids shows granum size-dependence, indicating that size of grana is one of the critical parameters in the regulation of the photochemical functions in the thylakoid.


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INTRODUCTION Photosynthesis is one of the most important photoinduced chemical reactions. In essence, it is an energy conversion process, i.e. the conversion of light into chemical reaction, and stores the photochemical products in the plant cells. Such a photoinduced chemical reaction occurs in chloroplasts of algae and plants. All the light-harvesting and some of the initial charge transfer processes of the photosynthesis are operative in thylakoid membranes inside the chloroplasts. The chlorophyll a, chlorophyll b, and carotenoids bound to the protein complexes essential for photosynthesis inside the thylakoids, which can be categorized into light harvesting complexes (LHC), photosystem I (PSI), and photosystem II (PSII), are in the proper orientation for the efficient interception and capture of light energy for photosynthetic function. Thylakoid membranes are simply separating lumen from stroma inside the chloroplasts, and they have two structural forms; i.e. stroma lamellae and granum. The stroma lamellae are the exposed thylakoid membranes without stacking arrangement, whereas the granum is a well-packed stacking thylakoid in the form of membrane discs with a typical diameter of a few hundreds of nanometers. There are a number of grana contained in a single chloroplasts, the three dimensional structure of which seems to play an important role for optimal photosynthesis.1-4 The unique and intriguing architecture of highly organized and regulated structure of the grana inside a chloroplast has received much attention. The three-dimensional arrangement of grana inside a chloroplast in intact leaves of plants has been evaluated by transmission- and scanningelectron as well as confocal microscopies.5-12 Notably, these microscopic techniques maintain the thylakoid, stroma, and grana network.1 The transmission- and scanning-electron microscopies with high spatial resolutions suggest that the arrangement of thylakoids and hence grana in


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chloroplasts is in such a manner so that the plants optimize photosynthetic efficiency to survive under fluctuating light source.8 These two microscopic techniques, however, require the thickness of specimen to be within a few hundreds of nanometers and fully dehydrated by chemical fixation, therefore they are not suitable for whole-leaf observation. On the other hand, although optical microscopy has lower spatial resolutions, it has been demonstrated to be useful to resolve living and functioning not only animal but also plant cells. One of them is fluorescence confocal microspectroscopy, which has become an important and powerful tool for structural, dynamic, and functional analysis of plant cells for decades.9-12 For example, by using super-resolution confocal fluorescence microscopy, Iwai et al. visualized thylakoid structures in a living cell of algae and revealed the dynamics of light-harvesting complexes.9 Such microspectroscopic study allows us to determine localization and distribution of pigments in vivo plant cells, such as Cyclotella meneghiniana and Phaeodactylum tricornutum.10 However, fluorescence microspectroscopic study of plant cells intrinsically depends on fluorophores. Because all the organelles do not contain fluorophores inside, it is natural and reasonable to stick fluorescence dyes on molecules specific to some target organelles for observations of their threedimensional arrangement in the cells.11 More importantly, it is known that PSI fluorescence is barely observable at room temperature, therefore, the detection and spectroscopic study with fluorescence microscopy is essentially difficult though Hasegawa et al. demonstrated that near-infrared laser light could excite antistokes fluorescence of PSI at room temperature.12 In studies on plant chloroplasts, changes in fluorescence spectra have been attributed to the state transitions of PSI and PSII as well as the processes of cell differentiation and/or assemblies of photosynthetic proteins.13-14 The fluorescence intensity is related to a product between fluorescence quantum yield and


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concentrations of the chlorophylls inside the grana and stroma lamellae, which are known to contain chlorophyll a and chlorophyll b with different ratios. The quantum yield can be probed by fluorescence-lifetime imaging microscopy, and but concentrations cannot be specifically resolved by fluorescence microscopy. Thus the grana and stroma lamellae in the subcellular organelle could be assessed by other confocal microscopic techniques. In this sense, scattering microscopy would be promising technique for mapping absorption spectra, as it will be described below, thus it can potentially resolve their molecular concentrations. We may note that though scattering microscopy would provide information on this aspect, however it cannot easily decipher excited state dynamics of the photosynthetic molecules. Considering that thylakoids are stacked and packed, forming a few hundred nm-sized grana, imaging and microspectroscopy will be useful to distinguish grana from stroma lamellae and starch in a chloroplast. In this sense, confocal Rayleigh scattering would be promising technique.15-17 The grana can be regarded as good light scatterers, therefore Rayleigh scattering microscopy could visualize their distribution in the plant cells. By combining this technique to confocal microscopy, out-of-focus light is blocked by pinhole, high scattering cross sections of the grana result in the Rayleigh scattering images and spectra with high contrast. Because intensity of the scattering light is linearly dependent on the incident light, intense laser pulses as the scattering light source would significantly increase the scattering intensity in the inhomogeneous system. From a view of light-harvesting system research, resonant light scattering is employed as an essential tool for studying chromophore aggregation.18-20 Furthermore, because light-scattering spectra are sensitive to absorption coefficients and the refractive indices of objects, the scattering efficiency spectrum of continuum light can intrinsically reflects the photon absorption of a particle with a particular geometrical size.21 Thus,


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this advantage makes the Rayleigh scattering microspectroscopy to be among inevitable microscopies, allowing one to evaluate the electronic properties of individual grana inside a chloroplast. In our study, we focus on Rayleigh scattering imaging and microspectroscopy with a polarized supercontinuum laser pulse train as the scattering light source which are sensitive to the granum size and alignment of chlorophyll and thylakoids. In this case, a polarized laser beam is focused and scanned on a sample to generate anisotropic scattering light. Thus, this allows us to evaluate anisotropic arrangement of the grana in a chloroplast of plant cells. Therefore this method is a promising tool to provide a deeper understanding of thylakoid organization in different granum sizes. The aim of this paper is to demonstrate that simultaneous acquisition of elastic scattering imaging and microspectroscopy, side by side, with a polarized laser pulse train as the scattering light source can be an innovative way to visually identify distribution and arrangement of grana in a chloroplast within an intact leaf. Here, we focus on a leaf of robust and rapidly growing Egaria densa (E. densa), which is also known as Anachris and among the most popular aquarium plants. By measuring the polarization-dependent scattering efficiency we may estimate the alignment of the pigments in a single granum inside the in vivo chloroplasts of E. densa. This novel technique provides observation on structural alignment of the pigments in different granum sizes, offering further evidence that the thylakoids have granum-size dependent and structural role in regulating the pigments bound to the protein complexes essential for photosynthesis.

EXPERIMENTAL SECTION


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Sample. We used a freshly detached leaf of E. densa. The leaf was cut in 1×3 mm2 and this specimen was sandwiched between two coverglass plates as a sample cell, and this sample cell was mounted and fixed on the sample stage of microscope. Rayleigh Scattering Imaging and Microspectroscopy. The optical setup in this work is shown in Figure 1. With this setup, we measure elastic scattering in the backward direction. Briefly, we employed a laser beam from a mode-locked Ti:sapphire laser (Spectra Physics; Tsunami) operated at 800 nm in fs pulse mode (80 fs; 80 MHz, 800 mW). The laser beam was directed into photonic crystal fiber (Crystal Fibre; NL-PL-750) to generate white supercontinuum light (λ=440‒760 nm, see Fig. S1).16 The supercontinuum laser pulse train then was attenuated with a ND filter to less than 10 mW and collimated by two positive lenses. As schematically shown in Fig. 1, after passing a spatial filter, the white supercontinuum pulse train was divided into two parts by a beam splitter. One part was directly directed onto the polychromator and it was considered as a reference beam. Another part of the supercontinuum beam was directed into an Olympus FV300 system with a pair of X-Y galvanometric mirrors to achieve raster scanning. The beam was subsequently introduced into an inverted microscope (Olympus; IX-71), and focused by an objective lens (Olympus; UPlanFl, 100×; NA 1.3) into the specimen mounted on the sample stage. The average laser power at the specimen is about a few mW. The back scattered light from the specimen were collected by the same objective, and it was sent back into an Olympus FV300 system. One half mirror inside FV300 directs the back scattered light into a photomultiplier tube (PMT, Hamamatsu, Japan) for imaging. The focal position of the supercontinuum pulse train is raster scanned by the galvanometric mirrors and the pixel dwell time is adjusted, so that the acquisition time for one X-Y image (256 × 256 pixels) is about 0.6 sec. By controlling the position of the objective lens along the z-axis automatically, the focal


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position for Z-direction was controlled inside the specimen. Once a X-Y image is obtained, we can select a specific position in the image and control the galvanometric mirror to obtain back scattered light from the position, then it is sent to a polychromator (Princeton Instruments; SpectraPro 2300i) with a 600 gr/mm grating and a cooled CCD camera (Princeton Instruments; PIXIS 400) for scattering spectrum detection instead of PMT by switching a mirror. By dividing the scattered light spectrum (Isca(λ)) by the incident scattering light source (Iin(λ)), the scattering efficiency (Ieff(λ)) was calculated for each type of chloroplast by using16

I eff ( λ ) = I sca ( λ ) I in ( λ )

(1)

Bright and Dark Field Microscopy. As shown in Figure 2, we also monitor the chloroplasts in the specimen by detecting bright or dark field images of the specimen around the focal spot area. For these purposes, we illuminated the sample cell by a white-light probe of halogen lamp (λ=380–750 nm) either the probe light is focused onto the sample cell through a conventional condenser lens (Olympus; IX2-LWUCD) or by using a dark-field condenser lens (Olympus; UDCW). The bright and dark-field images were collected in the forward direction by the same objective lens for confocal measurements and the transmitted light was passed through into a CCD camera (JAI; CV-A55 IR). Fluorescence Microscopy. In addition, we measured fluorescence spectrum of grana within the chloroplasts in the specimen. The fluorescence spectrum was detected at 620–740 nm by using polychromator (Princeton Instruments, USA) in the backward direction after photon excitation at 488 nm. In this case, continuum wave laser, instead of the supercontinuum laser pulse train was introduced into the Olympus FV300 system, and backward signal was directed into the polychromator. The procedure to record fluorescence spectrum is identical to that for Rayleigh


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scattering spectral measurement. Thus after taking a fluorescence X-Y image, we measure a fluorescence spectrum from a specific point in the image with an aid of the galvanometric mirror.

RESULTS AND DISCUSSION Figure 2 shows the specimen as well as bright and dark field images of the chloroplasts which are recognized in the microscope image as small and green structures within living cells inside the specimen. The green color comes from the chlorophylls, reflecting some of the light enriched in green wavelength. As can be seen in the images, individual chloroplasts inside a single cell can be outlined and they occupied cavities within each cell. Both bright and dark field images (Figure 2C-D) obviously indicate that the chloroplasts are not uniformly distributed inside the cell. We recall that each cell of green plants usually contains 10‒100 chloroplasts.22 Both the bright and dark field images show discrete spots of chloroplasts. By comparing the two images and plotting a line profile, it is clear that the chloroplasts observed in the bright field image are the same with those in the dark field image. However, the chloroplasts and cell wall are better identified in the dark field image. Using confocal Rayleigh scattering microscopy, it was possible to visualize the chloroplasts in a fresh leaf. As shown in Figure 3A, a backward scattering image with lateral resolution being 255 nm (See Fig. S2) demonstrates that the chloroplasts (a few µm in sizes) are stacked within the living cells besides the cell walls, consistent with bright and dark field images. The scattering image of the grana or stacked thylakoids inside the chloroplasts can be practically visualized by zooming in the image (Figure 3B) with grana appearing as multiple bright spots, showing the location of scattering particles randomly dispersed within each chloroplast. We consider that


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substantially high intensity of some bright spots in a single chloroplast shown in Figure 3A is due most probably to the stacking configuration of two or more grana along the Z-direction, and on the other hand much low intensity of bright spots is related to either small or out-of-focus grana. Such different intensity of bright spots due to stacking configuration and out-of-focus position was evaluated by shifting the focal position along the Z-direction. We may consider that mechanism of visualizing grana by Rayleigh scattering is primarily sensitive to absorption spectrum and refractive index of the grana. We may note that, in literature, grana within a chloroplast have also been visualized as bright spots by the fluorescence spectroscopy.23-24 In this sense, the mechanism for visualizing grana is based on fluorescence lifetime and quantum yield of PSII, where fluorescence lifetime and quantum yield are increased with the excitation light due to the famous closing of the reaction center.25 Such uneven distribution of thylakoids within a chloroplast has been known from various electron microscopy studies.1-4 The stacked thylakoids are further demonstrated to be interconnected in all directions by intergranal lamellae. It is interesting to notice from our finding that the grana, as well-packed thylakoids, can scatter the supercontinuum laser pulse train because of their strong light absorption in visible region and can be regarded as nanoparticles. This is in marked contrast to the stroma lamellae and starch in a chloroplast which are not good scatterers because of less absorption in visible, therefore both stroma lamellae and starch could not be traced from the scattering images. We may anticipate that the three-dimensional architecture for chloroplast consists of grana arranged in non-overlapping rows, in which thylakoids are arranged in an anisotropic manner. Such detailed organization of thylakoid networks in grana have been developed from transmission and scanning electron microscopic and electron tomography analyses of living


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cells.1, 6-7 The grana are further organized laterally in a non-regular arrangement interconnected with varying lengths by intergranal stroma lamellae in the chloroplast. This model has been widely established and accepted.4 We may recall that stroma lamellae also wind around grana forming a right-handed helix.2, 4 In this regard, we have tried to explore an evidence of the nonregular arrangement by measuring fluorescence of chloroplasts in the plant cells after 488-nm photon irradiation. Such an excitation results in fluorescence images and an emission band extending from 640 to 740 nm with a peak at 680 nm as shown in Figure 4 and 5, respectively. In this sense, we consider that the chlorophylls and carotenoid inside the thylakoid are responsible for the emission, and most of the thylakoid fluorescence should be emitted from chlorophyll a.26 Since the emission spectrum is apparently almost independent on the granum size across the chloroplast, we can expect that the emission likely results from the identical pigments in each granum, e.g. the abundant photoreceptive molecules of the chloroplasts inside the grana. The fluorescence image shows the space between the bright grana that likely correspond to stroma and stroma lamellae occupied in the chloroplasts. One can also see that when we focus on the image of a single granum, the fluorescence image provides poorer resolution than light scattering images. The poorer resolution fluorescence images are most probably due to short fluorescence lifetime of PSII reaction centers and lower fluorescence quantum yield of bound photoreceptive molecules in the grana, although the extinction absorption cross section at 488 nm by chlorophylls and carotenoid is sufficient to induce fluorescence emission.13-14 It has been described by Schreiber et al. that PSII may show roughly fluorescence quantum yield of 20% against the weak measurement light or under dark adaptation, and it may show even greater fluorescence quantum yield by the help of saturating light.26 Due to the same reasons, intergranal lamellae in the chloroplast are also unable to be resolved by the fluorescence imaging.


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For further quantitative analysis, owing to better resolution of scattering than fluorescence images under our experimental conditions, we estimated the sizes of the grana from the scattering images. Because the light scattering image of nanoparticles whose size is smaller than the wavelength of incident light can be considered as the convolution of the laser beam intensity profile and the particle size, the area of light scattering image from nanoparticles should reflect size of the particles. Thus, we extracted a line profile passing through the bright spots and we estimated the sizes of the grana (Figure 6A and B). Most of the grana image on the focal plane showed spherical shape and we took the average of at least three line profiles as the grana size. For some ellipsoidal grana images, we extracted the long axis for the line profile. But we should point out that the aspect ratios of all the ellipsoidal grana were less than 1.2. We analyzed a few hundreds of grana and we summarized the frequency versus the size distribution of grana in Figure 6C. The analysis demonstrated that the sizes of the grana were normally distributed within 180 to 450 nm with an average size of 270 nm. This average size and the size distribution is roughly consistent with that of PSII particles in spinach leaves (Spinacia oleracea) determined by freeze-fracture electron microscopy.27 By using Eq. (1) we have evaluated scattering efficiency spectrum of different sizes of granum. As shown in Figure 7 the spectra show an intense band around 450‒550 nm followed by several low intensity bands extended from 550 up to 700 nm. Those scattering efficiency spectra fairly resemble typical absorption spectra of porphyrins with the intense band being their Soret band, or absorption spectra of a mixture of porphyrins and carotenoids.26 If we consider that the scattering efficiency spectra reflect electronic structure of the photoreceptive molecules inside the thylakoids (and thus grana), the similarities between the scattering efficiency spectra of grana and absorption spectra of porphyrin and carotenoid derivatives may not be surprising, as in all


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eukaryotic photosynthetic organisms the most abundant photoreceptive molecules in the thylakoids are light-absorbing porphyrins and carotenoids bound to the proteins associated primarily with the photosynthesis. These chlorophyll and non-chlorophyll pigments absorb the light energy in the range from UV to the visible region, mainly in the red and blue parts of the spectrum. In order to explore the size-dependent scattering efficiency spectra, we evaluated the spectra of large number of grana. We found that the peak of the intense Soret band progressively shifted toward longer wavelength from 490 to 525 nm with the granum size as shown in Figure 8. We should emphasize that individual grana with comparable sizes show the similar scattering spectra and thus the absorption maximum of the Soret band, irrespective of their location at the center or in more peripheral surface of a chloroplast (see Figure 7). The red shift in the peak position means that energy of the Soret band is somewhat lower for larger granum size. This finding indicates that the photoreceptive molecules in thylakoids depend on the granum size but they are independent on position of the grana in the chloroplasts. This may indicate that the photoreceptive molecules inside the thylakoid have either an extended π-electron conjugation, different ionic states associated with metalation, or overlapping arrangement of photoreceptive molecular stacks each other in larger grana. In this sense, we should mention that optical properties of polymer films and nanocrystals of poly-1,6-di(N-carbazolyl)-2,4-hexadiyne or polydiacetylene are also size dependent, ascribed the effective conjugated length of the πelectron in the polymer backbones or to structural confinement of the nanocrystals.28 It is shown that the effective conjugation length of the organic materials is reduced and consequently their electronic band-gap is blue-shifted due to the higher concentration of structural defects in larger particle sizes. However, considering the photoreceptive molecules inside the thylakoids would


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not be transformed with the granum size, we proposed that the spectral shift of the Soret band is most likely due to more efficient overlapping of the photoreceptive molecules in larger thylakoid and/or higher packing structure of stacked thylakoids in larger grana. In this sense, the thylakoids may have thinner membranes or they are more tightly appressed and more hydrophobic in larger grana. Such the structural changes in thylakoids has been showed to be responsible to an absorption change and spectral shift in the Soret region.29 We may note that in some grana with intermediate sizes (250‒275 nm) as shown in Figure 8, the intense band has two peaks at 490 to 510 nm probably due to either strong intermolecular interactions or spectral overlap of the photoreceptive molecules in tightly appressed thylakoids. This assumption is supported by similar splitting of Soret band in extinction spectra reported in the case of porphyrin nanoparticles30 and self-assembly of chlorophyll a formation,31 suggesting two dimensional packing structure of molecules. On the other hand, similarity of the split band around 500 nm of the scattering efficiency spectra to absorption spectra of carotenoids should be pointed out.29 Because the scattering spectra of single grana show less intensities at Q band positions of porphyrins (around 650 nm), there is another possibility that the band splitting is due to light scattering mainly from carotenoids in the thylakoids. At this moment, we cannot conclude the origin of the band splitting. Further investigation to clarify the ratio of chemical components based on this microspectroscopic approach will make this question clear. The granum size-dependent spectral shift was further evaluated in this investigation by applying a linearly polarized pulse train as incident scattering light source (Fig. 9). It should be noted that the polarization measurement is an advantage of single particle spectroscopy under a microscope, as demonstrated by several groups in the study of single light-harvesting system.32-33 By evaluating the scattering efficiency spectra of a single granum at different polarization angles


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with respect to the laboratory axis, we found that the spectra essentially changed with the polarization angle, where maximum and minimum intensity of the Soret band was observed at polarization angle orthogonal to each other (Fig. 9A). This polarization-related spectroscopic signature may show that the packing structure of stacked piles of thylakoids in grana is anisotropic, in which one stacks on top of another. Here, we show that relative value of the maximum and minimum intensity of the Soret band (Fig. 9B) increases with granum size, as shown in Figure 9C. On the other hand, full width at half maximum (FWHM) of the plot in Fig. 9B decreases with granum size as shown in Figure 9D. These behaviors indicate further that the anisotropic packing structure of stacked thylakoids is amplified in larger grana, consistent with evidence the red-shifted Soret band of larger grana. Taking into account the three-dimensional model where the grana stacks have the expected cylindrical shape, measuring ∼500 nm in diameter and 130–160 nm in height,34-35 our finding indicates that the cylinder of stacked thylakoids is elongated with the size of grana. It suggests that thylakoid disks in a stack are straight and parallel without any out-of-plane bending or distortion,1, 4 and the plant cells are independently able to shape their thylakoid membranes in their chloroplasts into the elaborate grana stacks for light-harvesting.36 Such anisotropic arrangement and non-centrosymmetric structure of thylakoids in grana have been demonstrated by nonlinear optical properties of grana.5

CONCLUSION We show that Rayleigh scattering imaging and microspectroscopy is possible to image biological specimen of a live intact leaf of robust and rapidly growing E. densa, demonstrating


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that this technique is useful to visualize the organization of the thylakoids and hence grana inside chloroplasts. The scattering efficiency spectra of individual grana presented in this study fairly resemble cooperative absorption spectra of porphyrins and carotenoids, which are directly related to photoreceptive molecules of the chloroplasts essential for photosynthesis. On the basis of the light scattering image analysis, individual granum size was evaluated and the relationship between their spectra and size was clarified. The data further indicate anisotropic packing structure of stacked thylakoids is amplified in larger grana, suggesting that thylakoids are more likely stacked up in a straight direction and the stacked thylakoids are elongated with the size of grana. From a viewpoint of microscopy, this Rayleigh scattering confocal microspectroscopy allows us to obtain scattering efficiency which can be attributed to absorption spectral features of individual nano-objects with wide spectral range. In addition, it does not matter whether the objects show fluorescence or not. Thus this microscopy can be alternative method to study LHC at room temperature. Furthermore, it can be developed to time-resolved measurement by combining with pump-probe technique,17 which can potentially reveal the direct relationship between the granum size/position and the excited state dynamics. For example, photoprotective energy dissipation in plant chloroplasts, called nonphotochemical quenching, accompanies a reorganization of the pigment-protein complexes within the stacked grana thylakoids.37 Because the light scattering spectrum is very sensitive to the orientation of pigments in grana, timeresolved Rayleigh scattering microspectroscopy will be an alternative tool of fluorescence lifetime imaging microscopy to follow the orientation change during light adaptation at each granum level.

ASSOCIATED CONTENT


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The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Light intensity spectrum of the supercontinuum laser pulse train, and the spatial resolution determination of the confocal Rayleigh scattering microspectroscopic system by measuring individual gold nanoparticles.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (T.U.); [email protected] (A.U.); [email protected] (H.M.) Author Contributions T.U. and H.M. conceived and designed the experiment. L.-T.H. performed the optical measurements. P.-Y.H. maintained the lasers and optical microscopes. T.U. and A.U. analyzed data and wrote the manuscript with contributions from all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the MOE-ATU Project (National Chiao Tung University) and the Ministry of Science and Technology of Taiwan to H.M. (MOST 105-2811-M-009-022) and to


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T.U. (NSC 98-2113-M-009-013-MY2). Partial financial support from JSPS KAKENHI (Grant No. 16K21325) to T.U. is also acknowledged. The referee is greatly acknowledged for his/her critical and detailed comments as well as his/her suggestive discussions which are useful and helpful to improve this manuscript.


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FIGURE LEGENDS Figure 1. Schematic diagram of the experiment set-up for Rayleigh scattering measurement. For fluorescence measurement, the supercontinuum incident laser pulse train was replaced with 488nm continuum wave laser beam.

Figure 2. Images of (A) E. densa; (B) Sample specimen; (C) Bright field; and (D) Dark field of its plant cells.

Figure 3. Confocal Rayleigh scattering images of the plant cells of E. densa in backward direction; (A) without and (B) with 10× zoom. The zoomed area is indicated in (A) as a square. Spatial resolution in the lateral direction is estimated to about 255 nm.

Figure 4. Confocal fluorescence images of the plant cells of E. densa in backward direction after excitation at 488 nm; (A) without and (B) with 10× zoom in.

Figure 5. Representative florescence spectra of chloroplasts in the plant cells of E. densa. The spectra are taken from four different positions in the chloroplasts and overlapped.

Figure 6. (A) Rayleigh scattering image of a single chloroplast, (B) line intensity profile passing through a single granum, and (C) a histogram of granum size distribution. The single granum is


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indicated in the circle of panel (A). The direction of the line profile is shown in panel (A) as a dashed line. The line profile is fitted by a Gaussian function (solid). On the basis of the full width at half maximum (FWHM) obtained from the line profiles of single grana images, the size of grana was estimated and the distribution is summarized in (C).

Figure 7. (A) Rayleigh scattering image of chloroplasts and (B) scattering efficiency spectra of three grana indicated as A~C in panel (A). Each of the grana differed their sizes which were estimated by FWHM of a line intensity profiles obtained from the image in panel (A).

Figure 8. Peak wavelength of Soret band in scattering efficiency spectra of single grana. Each plot indicates individual granum as a function of granum size. Because light scattering spectra of single grana show either single or split peaks in their Soret bands, they are distinguished each other in the plots (single peak: circle; split peak: square).

Figure 9. (A) An example of unpolarized and polarized scattering efficiency spectra of single granum. The degrees indicate angle of polarizer. (B) Polarization plots of scattering intensities at 532 nm for the spectrum in panel (A). (C) Relative value of the maximum and minimum intensity of the Soret band under different incident polarization angles indicated as A and B in panel (B) as a function of granum size. (D) FWHM of the polarization plot peak band indicated in panel (B) as a function of granum size.


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Figure 1.


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 7


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Figure 8


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Figure 9


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Size-Dependent Optical Properties of Grana Inside Chloroplast of

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