Journal of Microscopy, Vol. 248, Pt 3 2012, pp. 234–244 doi: 10.1111/j.1365-2818.2012.03668.x Received 4 April 2012; accepted 28 August 2012

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two-photon laser scanning microscopy

A.H. RESHAK∗,† & C.-R. SHEUE‡ ∗School of Complex Systems, FFPW, CENAKVA, University of South Bohemia in CB, Nove Hrady 37333, Czech Republic †School of Material Engineering, Malaysia University of Perlis, P.O Box 77, d/a Pejabat Pos Besar, 01007 Kangar, Perlis, Malaysia ‡Department of Life Sciences, National Chung Hsing University, 250, Kuo Kuang Rd, Taichung 402, Taiwan

Key words. Chloroplast, multifunctional two-photon laser scanning microscopy (MF-2PLSM), second harmonic generation (SHG), Selaginella erythropus, two-photon excitation fluorescence (TPEF).

Summary SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation Background: Multifunctional two-photon laser scanning characteristic. microscopy provides attractive advantages over conventional two-photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic Introduction generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were Nonlinear optical effects, such as two-photon (Denk et al., achieved from the deep shade plant Selaginella erythropus. 1990) and three-photon (Wokosin et al., 1996; Maiti et al., Results: These measurements show that the S. erythropus 1997; Schrader et al., 1997; Tuer et al., 2008) fluorescence, leaves produce high SHG signals in both directions and significantly improve depth resolution and reduce the the SHG signals strongly depend on the laser’s status of background noise. Nonlinear optical techniques have been polarization and the orientation of the dipole moment in used to develop a new generation of optical microscopes with the molecules that interact with the laser light. The novelty novel capabilities. These new capabilities include the ability of this work is (1) uncovering the unusual structure of to use near-infrared light to induce absorption and enhance S. erythropus leaves, including diverse chloroplasts, various fluorescence from fluorophores that absorb in the ultraviolet cell types and micromophology, which are consistent with region. Other capabilities of nonlinear microscopes include observations from general electron microscopy; and (2) using improving spatial and temporal resolution without the use of the multifunctional two-photon laser scanning microscopy pinholes or slits for spatial filtering, improving signal strength by combining three platforms of laser scanning microscopy, for deeper penetration into thick and highly scattering tissue fluorescence microscopy, harmonic generation microscopy and confining photobleaching to the focal volume (Denk and polarizing microscopy for detecting the SHG signals in et al., 1990). The invention of nonlinear laser microscopy the forward and backward directions, as well as two photon has opened new opportunities for noninvasive examination of excitation fluorescence. the structure and functioning of living cells and tissues (Denk Conclusions: With the multifunctional two-photon laser et al., 1990). scanning microscopy, one can use noninvasive SHG imaging Among different multiphoton implementations (Zumbusch to reveal the true architecture of the sample, without et al., 1999; Zipfel et al., 2003), second harmonic generation photodamage or photobleaching, by utilizing the fact that the (SHG) imaging (Roth & Freund, 1980; Freund et al., 1986; Campagnola et al., 2001; Yeh et al., 2002; Campagnola Correspondence to: A. H. Reshak, Tel: +420 777729583; fax: +420–386 361255; & Lowe, 2003; Cox et al., 2003) is particularly suitable e-mail:[email protected];andC.R.Sheue,Tel/fax:+886422857395;e-mail: for investigating noncentrosymmetric structures. SHG is a [email protected] nonlinear optical process that occurs only at the focal point of

C 2012 The Authors Journal of Microscopy C 2012 Royal Microscopical Society SECOND HARMONIC GENERATION IMAGING 235 a laser beam (Shen, 1989). The application of SHG imaging is investigated by means of the multifunctional-two-photon of cellular structure and functioning is quite new and notable laser scanning microscopy (MF-2PLSM), which the first (Campagnola & Loew, 2003). Advances in mode-locked lasers author established by combining three platforms of laser [instead of a continuous wave, mode-locked lasers, which emit scanning microscopy: fluorescence microscopy, harmonic short pulses in the range of nanoseconds to femtoseconds (fs)] generation microscopy and polarizing microscopy. MF- makes SHG imaging of cells possible, because lower intensities 2PLSM provides attractive advantages over conventional can be used (Reshak et al., 2009). Using chiral chromophores, fluorescence microscopy for revealing the true architecture chiral SHG imaging can be applied to otherwise impossible of the samples that can not produce autofluorescence without symmetric structures (Yan et al., 2006). labelling or staining, which might induce undesirable effects Second harmonic imaging microscopy (SHIM) is based on a in the living cell. Reconstruction of complementary images by nonlinear optical effect known as SHG (Barzda et al., 2004; eliminatingtheangledependenceofimages,whenusinglinear Barzda et al., 2005; Greenhalgh et al., 2006). SHIM has polarized laser, helps maximize the SHG signals and hence been established as a viable microscope imaging contrast improves the brightness and the sharpness of the features in mechanism for visualization of cell and tissue structure and SHG images of samples. This technique will provide biologists function. SHIM using SHG as a probe is shown to produce and medical researchers another useful visualization tool for high-resolution images of transparent biological specimens exploring the nature of living cells. (Campagnola & Loew, 2003). A second harmonic microscope The study organism, S. erythropus, is an unusual plant obtains contrasts from variations in a specimen’s ability growing in the low light understory of tropical rain forests. A to generate second harmonic light from the incident light giant chloroplast, termed a bizonoplast, was first discovered in whereasaconventionalopticalmicroscopeobtainsitscontrast this plant (Sheue et al., 2007). The bizonoplast is characterized by detecting variations in optical density, path length or by unique dimorphic ultrastructure differentiating the refractive index of the specimen. SHG requires intense laser chloroplast into upper and lower zones. However, the light to pass through a material with a noncentrosymmetric leaves (viz. microphyll) of S. erythropus also contain typical molecular structure (Reshak et al., 2009). Second harmonic chloroplasts. Novel patterns of silica bodies on leaf surface light emerging from SHG material is exactly half the of this plant have also been observed (Sheue et al., 2006). wavelength (frequency doubled) of the light entering the Baseline studies of the leaf structure of this plant from general material (Reshak et al., 2009). The alternative technique, electron microscopy contrast with MF-2PLSM, revealing the two-photon-excited fluorescence (TPEF) is also a two-photon advantages of these new nonlinear techniques to better process. TPEF involves some energy loss during relaxation understand this deep shade plant noninvasively. from the excited state, whereas SHG is energy conserving. Advances in the developments of SHIM have provided Material and methods researchers with novel means by which noninvasive visualization of nonbiological and biological specimens can be Laser sources and imaging system achieved with high penetration and high spatial resolution, and is known to leave no energy deposition on the interacting The schematic of the MF-2PLSM is shown in Figure 1. This matter because of SHIM’s virtual energy conservation MF-2PLSM consists of an inverted i-mic 2 microscope (Till- characteristic (Gao et al., 2006). That is, the emitted SHG Photonics, Grafelfing, Germany), equipped with Ti:sapphire photon energy is the same as the total absorbed excitation femtosecond laser with a tuning range of 690–820 nm. The photonenergy.Theinhomogeneityinherenttomostbiological laser is a Tsunami 3941-M3B pumped by a Millennia-V, specimen, and in particular, to the internal structure of 5W solid-state pump laser (Spectra-Physics, Mountain View, various cells, leads to high quality SHG images without CA, USA). The Tsunami laser was used to generate linearly any preconditioning such as labelling or staining that might polarized pulses at 810 nm, 20 mW and 100 fs pulse width induce undesirable effects in the living cell (Reshak, 2009). at frequency of 80 MHz, for fluorescence excitation and SHG. Historically, resolution in fluorescence optical microscopy has Thus, in general to maximize the signal (fluorescence emission been limited by the Rayleigh criterion. The Rayleigh criterion and SHG), short pulses should be used and average laser states that two images are just resolved when the principal power should be kept low to prevent heating of the sample maximum (of the Fraunhofer diffraction pattern) of one image as well as unwanted one-photon absorption and to reduce coincides with the first minimum of the other (Born & Wolf, the risk of highly nonlinear photodamage (Denk et al., 1995). 1980). Techniques with better resolution than the Rayleigh A beam expander was used to fill the back aperture of the criterion have recently been established, among which is objective and λ/2 plate was used to control or maximize harmonic excitation light microscopy (Frohn et al., 2000). the status of the laser’s polarization at the sample. The The novelty of this work is the application of these excitation light was directed onto a pair of galvanometer techniques to reveal the structure of plant tissue. In particular, XY scanners (Yanus; Till-Photonics). The scanned excitation for the first time the deep shade plant Selaginella erythropus light was focused onto the sample through the microscope

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objective to scan the sample in the x–y direction at the focal plane. The stage of the microscope is driven by a computer controlled motor to take the sample to different z positions following each x–y scan. The scanning mirrors are metal coated (silver) with a good thermal resistance (Diaspro, 2001). Further components from the set-up in Figure 1 are: the dichroic mirrors [DM-1: Q565LP for TPEF (for the materials which produce autofluorescence) (Fig. 1b]; emission filter EF-1: red glass 665 nm; DM-2: Omega 475DCLP for SHG (Fig. 1c); interference blue emission filter EF-2 (405 nm; Fig. 1d); photomultipliers: Hamamatsu R6357; objective 1: Olympus uplanApo/IR 60×/1.20 water immersion; objective 2: Zeiss 40×/1.2W korr or Olympus uplanFLN 10×/0.3). The Laser power was maintained to be 20 mW at the Tsunami aperture and 5 mW at the sample. Additional infrared beam block filters BF (HQ700SP-2p 58398) were placed in front of each photomultiplier to ensure that illumination light was effectively suppressed and only TPEF or SHG signals were recorded. For SHG imaging, optical filtering is achieved with an interference filter centred on the expected SHG frequency (Fig. 1d) configurations of the photomultipliers were identical for both SHG and TPEF imaging. This set-up will enable the simultaneous measurement of SHG in the forward and backward directions as well as TPEF (Barzda et al., 2004). The signals from the photomultipliers are reconstructed by a computerintoimages.Imageswereobtainedinstacksstepping along the z-axis with 0.5 μm steps. Preliminary imaging of the sample has been performed with a scan rate of 0.25 s−1 (512 × 512 pixels) and signal-to-noise ratio is about 20 dB. The lateral resolution is about 270 nm and the axial resolution is 973 nm using Olympus uplanApo/IR 60×/1.20 water immersion objective. The microscopy is controlled via a standard high-end Pentium-4 PC and linked to the electronic control system via an ultrafast interface.

Material The material used here is S. erythropus, a deep shade plant native to South America. This plant was originally collected from Singapore Botanic Gardens and grown in the laboratory in a deep shade environment. General electron microscopy was applied to semithin sections of a leaf prepared by standard TEM methods (Sheue et al., 2007). In addition, a tabletop microscope (TM3000, Hitachi, Japan) gave leaf surface images. This plant was moved to a dark location for two weeks before the investigation of MF-2PLSM to eliminate starch grains from the chloroplasts. To apply MF-2PLSM, a leaf Fig. 1. Design of the MF-TPSLM in this study. (a) The experimental set- was detached with watchmakers forceps from a darkened part up using two objectives for collecting the forward SHG signals (objective Zeiss40×/1.2waterimmersionorobjectiveOlympusuplanFLN10×/0.3) of the plant. The leaf was mounted between two cover slips and the backward SHG signals (objective Olympus uplanApo/IR 60×/1.2 in water and the edges of the smaller cover slip were sealed water immersion or objective Olympus uplanFLN 10×/0.3). The third to the lower larger cover slip by means of nail varnish. The objective is for collecting the TPEF (objective Olympus uplanApo/IR paired cover slips were placed on the stage of a Till-Photonics 60×/1.2 water immersion). (b) Dichroic mirror Q565LP for TPEF. (c) microscope and illuminated with a Titanium sapphire laser Dichroic mirror Omega 475CLP for SHG. (d) Emission filter 1 for SHG. at 810 nm (linearly polarized laser), 5 mW and 100 fs pulse

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Fig. 2. Morphology, leaf structure and chloroplast ultrastructure of Selaginella erythropus. (a) Shoots showing the anisophyllous and dorsiventral structure with two rows of small dorsal leaves and two rows of large ventral leaves on each branch of its stem. (b) A transverse section of a branch showing the arrangement of leaves around the central stem axis with dorsal leaves (on the top) and ventral leaves (below). Here we apply the terms ‘ventral side’ to the lower surfaces and ‘dorsal side’ to the upper surfaces of both types of leaf. (c) Silica bodies on the ventral side of a ventral leaf. (d) Ventral leaf cross section near the vein area showing the internal leaf structure, chloroplasts and silica bodies (arrows). (e) TEM view of a giant cup-shaped chloroplast (bizonoplast) located in a dorsal epidermal cell and characterized by a unique dimorphic ultrastructure differentiating the chloroplast into upperand lower zones. (f) TEM view of a typical chloroplast in a ventral epidermal cell. Bp, bizonoplast; Cm, chloroplast of a mesophyll cell; Cv, chloroplast ofa ventral epidermal cell; CW, cell wall; DE, dorsal epidermal cell; DL, dorsal leaf; DLDS, dorsal leaf dorsal side; DLVS, dorsal leaf ventral side; LZ, lower zone; M, mesophyll; Mi, mitochondrion; S, starch grain; St, stoma; UZ, upper zone; V, vein; Va, Vacuole, VE, ventral epidermal cell; VL, ventral leaf; VLDS, ventral leaf dorsal side; VLVS, ventral leaf ventral side. width. The objectives were aligned relative to one another and the axis (viz. stem) is in the middle and the leaf structure of both focused on the sample. A set of images was captured. types is basically the same relative to the vertical direction, here we apply the terms ‘ventral side’ to the lower surfaces and ‘dorsal side’ to the upper surfaces of both types of leaf Results rather than the common terms ‘abaxial and adaxial sides’, Selaginella erythropus is anisophyllous, with two rows of small which with this unique foliar arrangement are not helpful. dorsal leaves and two rows of large ventral leaves on each The dorsal sides of both dorsal and ventral leaves are green branch of its stem (Fig. 2a). The arrangement of these leaves is (Fig. 2b). The ventral side of the dorsal leaf, which cannot be prominently dorsiventral, with a stem located between dorsal easily viewed from either the dorsal or ventral surface of the leaves on the top and ventral leaves beneath (Fig. 2b). Because shoot,isgreenexceptforaredmargin,whereastheventralside

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Fig. 3. Backward SHG signal of the ventral side of the dorsal leaf from randomly orientated linearly polarized laser. (a) Before adjusting the direction of polarization of the linearly polarized laser. (b) After maximally orientating the direction of the linearly polarized laser to the orientation of the dipole moment in the molecules. The backward SHG signal was collected by the objective Olympus uplanFLN 10×/0.3. The double arrow shows the direction of the polarization of the 810 nm laser beam. The procedure in Figure 3(b) for orientating the polarization of the laser was applied also to produce Figs 4–6. V, vein. of the ventral leaf is deep red. Silica bodies appear as conical objective Olympus uplanFLN 10×/0.3. Figure 3(a) shows the protrusions from the epidermal cell walls on both surfaces of weak signal of the SHG before the orientation of the laser’s dorsal and ventral leaves as previously reported by Sheue et al. polarization parallel to the orientation of the dipole moment (2006). Silica bodies forming a single row on a single ventral in the molecules; then after slowly changing the polarization’s epidermal cell of ventral leaves are the most evident silica body direction of the laser beam, the SHG signals significantly pattern (Fig. 2c). Stomata are aggregated in a band along the increased to reach the maximum value, as it is illustrated vein on the dorsal side of dorsal leaves and the ventral side of by Figure 3(b). ventral leaves (Fig. 2d). Figures 4 and 5 show the simultaneously acquired forward Dorsal and ventral leaves of S. erythropus are six cells thick and backward SHG images of the dorsal and ventral surfaces of in the vein region, with leaf thickness gradually reduced a dorsal leaf. The forward SHG signal was collected with a Zeiss to two layers (the upper and lower epidermis) towards 40×/1.2 water immersion objective and the backward SHG the margin (Fig. 2d). The outer tangential cell wall of signal was collected using the objective Olympus uplanApo/IR ventral epidermal cells is very thick with multiple layered 60×/1.2 water immersion. These figures of the forward ultrastructure (Figs 2d and e). Chloroplasts are found in (Figs 4a, c and 5a, c, e) and backward images (Figs 4b, d dorsal epidermal cells, mesophylls and ventral epidermis, and 5b, d, f) are almost identical except that the backward including guard cells in leaves of S. erythropus. However, images usually have slightly higher contrast. Figure 4 shows the size and number of chloroplasts vary between these the images of the dorsal epidermal cells with a stomata band tissues (Table 1). Bizonoplasts, giant cup-shaped unique along the middle part. The area of this stomata band is slightly chloroplasts with dimorphic ultrastructural organization in curved, leading to different focal planes under a microscope. a single chloroplast, are located in dorsal epidermal cells: The outlines of the dorsal epidermal cells, stomata and guard the upper zone is occupied by numerous layers of two to cells surrounding stomata can be recognized easily with SHG four stacked thylakoid membranes, whereas the lower zone signals. The bizonoplasts in dorsal epidermal cells are revealed contains both unstacked stromal thylakoids and thylakoid as much bigger than the chloroplasts in the mesophyll cells lamellae stacked in normal grana structures oriented in and guard cells (Figs 4a and b). Compared to a single giant different directions (Fig. 2e). The chloroplasts in other tissues chloroplast in a dorsal epidermal cell, there are three to five (suchasmesophyll,ventralepidermis)arenormalchloroplasts chloroplasts per mesophyll cell and four chloroplasts per guard and are smaller. These observations viewed by LM, SEM and cell (confirmed by confocal scanning light microscopy, data TEM provide a substantial basis of comparison for the results not shown). Scanning to a deeper position of these dorsal from SHG signals. epidermal cells reveals numerous vacuole-like vesicles in each Figure 3 shows the backward direction SHG signal of the cell (Figs 4c and d). Whether these signals are derived from ventral side of the dorsal leaf before (Fig. 3a) and after (Fig. 3b) vacuoles or other organelles needs further investigation. maximizing the polarization of the linearly polarized laser in SHG images from the ventral epidermis show that its outer the orientation of the dipole moment in the molecules. The tangential cell walls have very strong signals with multiple orientation of the laser’s polarization is illustrated by double layered dark curve patterns (Figs 5a and b). Compared to arrows in Figure 3. These images were collected using the isodiametric dorsal epidermal cells, ventral epidermal cells are

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Fig. 4. SHG signals of the dorsal side of the dorsal leaf (polarization as in Fig. 3b). (a, c) Forward SHG signals collected by the objective Zeiss 40×/1.2, water immersion, these are slightly different focal planes. (b, d) Backward SHG signals collected by the objective Olympus uplanApo/IR 60×/1.2, water immersion. These figures show giant bizonoplasts in the dorsal epidermal cells, and smaller chloroplasts in mesophyll cells and guard cells. Bp, bizonoplast; Cg, chloroplast in a guard cell; Cm, chloroplast in a mesophyll cell; Cv, chloroplast in a ventral epidermal cell; DE, dorsal epidermal cell; St, stoma. These figures are slightly different focal planes. much elongated with smaller bead-like chloroplasts arranged (Figs 6e and f) and dorsal leaves (Figs 4 and 5) are similar in as chains (Figs 5c and d). There are three to five disc-shaped the patterns of size and arrangements of chloroplasts in dorsal chloroplasts in a mesophyll cell, with median size (Figs 5e epidermal cells, mesophyll cells and ventral epidermal cells. and f). Because the simultaneously acquired forward and Discussion backward SHG images are very similar (see supplementary figures), here we show only the backward SHG images This study reveals high SHG signals in the leaves of of the dorsal and ventral surfaces of the ventral leaf S. erythropus originating from micromophology, cell walls, (Fig. 6). These images were collected using the objective cell contents and chloroplasts. Various categories of size and Olympus uplanApo/IR 60×/1.2 with water immersion. The number of chloroplasts can be recognized from the leaves isodiametric dorsal epidermal cells (Fig. 6a) and oblong of S. erythropus (Table 1), in strong agreement with the mesophyll cells (Fig. 6b) shown in the top view in a ventral leaf observations of Sheue et al. (2007). These diverse chloroplasts aresimilartothoseobservedinadorsalleaf.Silicabodiesonthe include (1) large cuplike chloroplasts, bizonoplasts, in the ventral side of a ventral leaf also emit strong signals (Figure 6d) dorsal epidermal cells; (2) disk-shaped chloroplasts in the matching the results observed by SEM in Figure 2(c). The mesophyll; (3) elongated or beadlike chloroplasts arranged smallest chloroplasts in the leaves of S. erythropus were as a chain in the elongated, ventral epidermal cells; observed in the trichomes of the leaf margin near the basal part (4) trichome chloroplasts; and (5) stomatal chloroplasts. In (Figs 6c and e), note that trichomes along the leaf margin have terms of ultrastructure, only the bizonoplasts have dimorphic smaller chloroplasts (arrows) see Figure 6(c). Ventral leaves ultrastructure: the upper zone is occupied by numerous layers

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Fig. 5. SHG signals of the ventral side of the dorsal leaf (polarization as in Fig. 3b). (a, c, e) Forward SHG signals collected by the objective Zeiss 40×/1.2, water immersion. (b, d, f) Backward SHG signals collected by the objective Olympus uplanApo/IR 60×/1.2, water immersion. (a, b) The top of ventral epidermal cells with outer tangential cell walls and some chloroplasts. (c, d) Chloroplasts in ventral epidermal cells, beadlike, arranged in chains. (e, f) Chloroplasts in mesophyll cells, which are larger than those in ventral epidermal cells. Cm, chloroplast in a mesophyll cell; Cv, chloroplast ina ventral epidermal cell; CW, cell wall; M, mesophyll; VE, ventral epidermal cell. of two to four stacked thylakoid membranes whereas the mingled together. These features of chloroplasts observed lower zone contains both unstacked stromal thylakoids and from S. erythropus are consistent with previous findings that thylakoid lamellae stacked in normal grana. The other types many shade plants have large chloroplasts with numerous of chloroplasts in the leaves of S. erythropus are typical thylakoids per granum (Nasrulhaq-Boyce & Duckett, 1991; chloroplasts with grana and stoma thylakoid membranes Sarafis, 1998). The SHG signal is not as effective as TEM

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Fig. 6. Backward SHG signals of both sides of the ventral leaf collected by the objective Olympus uplanApo/IR 60×/1.2, water immersion (polarization as in Fig. 3b). (a) Isodiametric dorsal epidermal cells. (b) Oblong mesophyll cells near leaf tip. (c) Lower magnification of the ventral side near middle and basal parts showing abundant chloroplasts in tissues. Note that trichomes along the leaf margin have smaller chloroplasts (arrows). (d) Ventral epidermal cells near vein area with stomata. Silica bodies can be observed on the elongated ventral epidermal cells, but not on the stomatal band along the vein. There are four chloroplasts in each guard cell. (e) The area near the basal part of the leaf margin with trichomes (labelled with T). Chloroplasts in mesophyll cells, ventral epidermal cells and trichomes can be distinguished by size. (f) The beadlike chloroplasts arranged as chains in ventral epidermal cells are smaller than those in mesophyll cells. Cg, chloroplast in a guard cell; Cm, chloroplast in a mesophyll cell; Ct, chloroplast in a trichome; Cv, chloroplast in a ventral epidermal cell; DE, dorsal epidermal cell; M, mesophyll cell; Si, silica bodies; St, stoma; T, trichome; VE, ventral epidermal cell. in differentiating the upper zone and lower zone of a variations, most likely originating from grana, and the stacked bizonoplast, but it provides strong signals with information on regions of the thylakoid membranes (Garab et al., 2005). arrangement, shape and size of the five types of chloroplasts. The birefringence is important in fulfilling phase-matching The chloroplasts exhibit strong birefringence with large local conditions (Boyd, 1992; Reshak et al., 2008; Reshak, 2009).

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Table 1. Chloroplast diversity in microphyll of Selaginella erythropus. that interact with the laser beam. It is therefore advantageous to control the laser’s status of polarization, to maximize the Chloroplast no. per Average length SHG signals. μ = Cell type cell, shape and type ( m; N 10) Our results support the contention that the collecting Dorsal epidermal cell 1, cup, bizonoplast 26.7 ± 3.5 efficiency of SHG signals is highly dependent on the numerical Mesophyll cell 4∼6, disc, normal chloroplast 8.1 ± 1.5 aperture of the objective (Han et al., 2005; Cox et al., 2004; Ventral epidermal cell4∼9, beadlike, normal chloroplast 7.0 ± 1.0 Reshak et al., 2009). Higher values of numerical aperture Guard cell 4, oval, normal chloroplast 8.4 ± 1.1 with immersion medium allow increasingly oblique rays to Trichome 0∼13, disk, normal chloroplast 5.3 ± 0.6 enter the objective front lens, by capturing higher order of diffraction rays from the samples, producing a more highly resolved image (Reshak et al., 2009). The strength of the SHG The birefringence is the difference between the extraordinary signals significantly depends on the numerical aperture and and ordinary refraction indices. Generally, materials show the immersion medium of the objective. Also, it is strongly high birefringence (a considerable anisotropy in the linear dependent on the polarization direction of the laser beam. The optical susceptibility) that favours an important quantity sample will produce a strong SH signal when the polarization in second-order susceptibility (determining SHG) because of direction of the linearly polarized laser is parallel to the better fulfilling of phase-matching conditions, determined by orientation of the dipole moment in the molecules. birefringence (Reshak et al., 2008). SHG is very efficiently SH imaging is especially helpful for biological studies generated in chloroplasts (Chu et al., 2001). Chloroplasts of living samples. Acquiring fluorescence images with in celery showed a signal in the SHG image, which did conventional microscopy leads to photobleaching and not colocalize with the autofluorescence of the chlorophyll. photodamage, whereas the SH imaging process does not. Crystalline starch in starch grains is typically organized with Because the SHG does not use an absorptive process, the the crystallites in a radial fashion, yielding a characteristic intense laser field induces a nonlinear polarization in the cross image in polarized light (Clowes & Juniper, 1968). molecules resulting in the production of coherent waves, twice This in turn means that SHG image will be orientation the incident frequency. Moreover the SHG image results from dependent (Cox et al., 2004). The significant SHG seen in a few femtoseconds, and is energy conserving process. This is biological materials arises from low local symmetry and the another advantage of the SH imaging when one needs to work large nonlinear coefficient typical for biological molecules and with sensitive samples. Thereby, one can investigate the true structures (Lukins et al., 2003; Helmchen & Denk, 2005). architecture of the sensitive samples. The chloroplasts containing starch grains (Chu et al., 2001), which are strong sources of SHG signals. In this measurement, Conclusions the plant was kept in the dark for approximately 2 weeks to eliminate the starch. From above, one can conclude that This is the first time the deep shade plant S. erythropus has the origin of the high amount of SHG signals which comes been investigated by means of the MF-2PLSM established from S. erythropus leaves is attributed to the unusually large by combining three platforms of laser scanning microscopy. chloroplasts (bizonoplasts) and various categories of size and The MF-2PLSM offers several advantages for uncovering the number of chloroplasts with numerous and thick unusual true architecture of the sample and enables simultaneous thylakoid membranes, which are very strong sources of SHG measurement of the SHG signals in the forward and backward signals. In this study, the chloroplasts in trichomes are the directions. The leaves of S. erythropus produce very strong smallest chloroplasts with relatively weaker SHG signals than SHG signals that are attributed to various categories of the other chloroplasts in this plant. This result is consistent size and number of chloroplasts with numerous thylakoid with the observation of trichome chloroplasts examined membranes. Moreover, the leaves are multilayered providing by TEM (data not shown). The chloroplasts in trichomes another reason for the strong SHG signals, which accumulate have limited and poorly developed thylakoid membranes. from these layers. Cell wall, cell content and big silica In addition to the abundant SHG signals derived from bodies also provide signals. This measurement provides chloroplasts,somecellcontents,silicabodiesandcellwallsalso noninvasive, effective and informative images similar to displayed strong SHG signals. However, we do not know which paradermal sections of the leaf but without the disadvantages structure causes the curve pattern of SHG signals around of photobleaching and photodamage. ventral epidermal cells in Figures 5(a) and (b). Further study In summary, the SHG signals strongly depends on two is needed. objects: the first object is the microscope – the laser’s status As SHG was established by earlier works (Stoller et al., 2002; of polarization and the numerical aperture of the objective; Lukinsetal.,2003;Reshak2009;Reshaketal.,2009),theSHG and the second object is the biological materials – the signal strongly depends on the laser’s status of polarization structure of the materials whether if it is homogenous or and the orientation of the dipole moment in the molecules not, or centrosymmetric or non-centrosymmetric, and the

C 2012 The Authors Journal of Microscopy C 2012 Royal Microscopical Society, 248, 234–244 SECOND HARMONIC GENERATION IMAGING 243 orientation of the dipole moment in the molecules that interact Frohn, J.T., Knapp, H.F. & Stemmer, A. (2000) True optical resolution with the laser beam. This new emerging microscopy shows beyond the Rayleigh limit achieved by standing wave illumination. high potential for the study of living samples in biological and Proc. Natl. Acad. Sci USA 97, 7232–7236. medical research. Garab, G., Galajda, P., Pomozi, I., Finzi, L., Praznovszky, T., Ormos, P. & van Amerongen, H. (2005) Alignment of biological microparticles by a polarized laser beam. Eur. Biophys. J. 34, 335–343. Acknowledgements Gao, L., Jin, L.X.P., Xu, J.W.Y., Ma, H. & Chen, D. (2006) Reconstruction We would like to thank Prof. V. Sarafis and Singapore of complementary images in second harmonic generation microscopy. Botanic Gardens for providing the plants, Prof. P. Chesson Opt. 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