, Volume 252, Issue 5, pp 1231–1240 | Cite as

Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues

  • Yoko Mizuta
  • Daisuke Kurihara
  • Tetsuya Higashiyama
Original Article


In vivo imaging of living organisms is an important tool to investigate biological phenomena. Two-photon excitation microscopy (2PEM) is a laser-scanning microscopy that provides noninvasive, deep imaging in living organisms based on the principle of multiphoton excitation. However, application of 2PEM to plant tissues has not been fully developed, as plant-specific autofluorescence, optically dense tissues, and multiple light-scattering structures diminish the clarity of imaging. In this study, the advantages of 2PEM were identified for deep imaging of living and intact Arabidopsis thaliana tissues. When compared to single-photon imaging, near-infrared 2PEM, especially at 1000 nm, reduced chloroplast autofluorescence; autofluorescence also decreased in leaves, roots, pistils, and pollen grains. For clear and deep imaging, longer excitation wavelengths using the orange fluorescent proteins (FPs) TagRFP and tdTomato gave better results than with other colors. 2PEM at 980 nm also provided multicolor imaging by simultaneous excitation, and the combination of suitable FPs and excitation wavelengths allowed deep imaging of intact cells in root tips and pistils. Our results demonstrated the importance of choosing both suitable FPs and excitation wavelengths for clear two-photon imaging. Further advances in in vivo analysis using 2PEM will facilitate more extensive studies in the plant biological sciences.


Two-photon excitation microscopy (2PEM) Deep imaging Live-cell analysis Autofluorescence Multicolor Arabidopsis thaliana 


Recent advances in imaging technique using various fluorescence probes have revealed dynamic events at the subcellular level. Green fluorescent protein (GFP, Shimomura et al. 1962) and various GFP-like fluorescent proteins (FPs) have been widely used in molecular biology studies. In the late 1980s, the confocal microscope (a fluorescent microscope) was developed (Brakenhoff et al. 1985; White et al. 1987) in which a confocal pinhole is used to eliminate the fluorescence from non-focal planes and produce thin (<1 μm) and high-spatial-resolution images (Pawley 2006). Nevertheless, confocal imaging has some limitations, e.g., photobleaching of the fluorophore and phototoxicity to specimens. This problem has occurred via excitation in not only the focal plane, but also above and below the focal planes (Benninger and Piston 2013). In addition, confocal microscopy is less suitable for deep tissue analysis because the signal is degraded due to scattering or absorption from excitation and emission (Centonze and White 1998).

Two-photon excitation microscopy (2PEM) is laser-scanning microscopy using multiphoton excitation processes (Helmchen and Denk 2005; Benninger and Piston 2013). Multiphoton processes offer important advantages for physiological studies in living organisms. For example, out-of-focus photodamage and photobleaching are eliminated, as the multiphoton effect is spatially confined to the femtoliter within the focal plane (König 2000; So et al. 2000). Additionally, 2PEM uses the femtosecond near-infrared pulse laser, which achieves deeper penetration. Thus, 2PEM is a useful tool for performing less-invasive studies (Benninger and Piston 2013) and allows cellular imaging of several hundred to thousand microns inside living organisms (Centonze and White 1998; Helmchen and Denk 2005).

In the animal sciences, 2PEM has been used in a wide range of imaging studies, including those involving the lymphatic organ (Cahalan et al. 2003; Bousso and Robey 2004), kidney (Molitoris and Sandoval 2005), heart (Rubart 2004), skin (Laiho et al. 2005), and brain (Denk and Svoboda 1997; Helmchen and Denk 2002) tissues. Additionally, Kawakami et al. (2013) succeeded in visualizing cerebral cortex layers deeper than 1 mm in a living mouse. Numerous applications of 2PEM in animal tissues have been reported, whereas only a few studies have described its application to plants, although 2PEM technology has been used in Arabidopsis (Tirlapur and König 2001a; Feijó and Moreno 2004; Cheung et al. 2010), waterweed (Tirlapur and König 2001b), and tobacco (Schwille et al. 1999). Cheung et al. (2010) attempted to visualize the pollen tube (PT) growth in a living pistil by using 2PEM with GFP-labeled PTs, but imaging was only successful 120 μm inside the pistils. Plant tissues, such as those in chlorophyll, lignin, cellulose, and phenols (Müller et al. 2013), contain a variety of autofluorescent compounds (Rost 1995) that are causally related to plant autofluorescence and optically dense tissues containing multiple light-scattering structures. Thus, imaging of plant tissue is difficult, even at a depth of just several hundred microns (Feijó and Moreno 2004).

In this study, we analyzed proper excitation wavelengths and FPs for 2PEM in intact Arabidopsis tissues. We demonstrated the advantages of orange FPs with longer wavelength excitation to reduce plant autofluorescence, with 2PEM providing deep and multicolor imaging of Arabidopsis tissues in vivo.

Materials and methods

Plant materials and growing conditions

The Arabidopsis ecotype Columbia (Col-0) was used as the wild-type (WT) plant. We also used transgenic plants carrying LAT52p::FPs (FP: mTFP1, sGFP, Venus, TagRFP, and mRFP), RPS5Ap::tdTomato-LTI6b, GFP-mTalin (Oikawa et al. 2003; Kadota et al. 2009), RPS5Ap::H2B-sGFP (Maruyama et al. 2013), RPS5Ap::H2B-tdTomato (Adachi et al. 2011), and FGR8.0 (Völz et al. 2013). Transformants of LAT52p::FPs and RPS5Ap::tdTomato-LTI6b were prepared as mentioned below. Seeds were germinated on agarose plates at 22 °C under 24-h light. Fourteen-day-old seedlings were transferred to a mixture of Metro-Mix 350 soil (Sun Gro Horticulture, Agawam, MA, USA) and potting compost; seedlings were grown at 22 °C under 24-h light or long-day conditions (16-h light/8-h dark).


The pollen-specific LAT52 promoter (Twell et al. 1989) was used to express FPs in the PT. The complementary DNA (cDNA) of each FP was amplified by polymerase chain reaction (PCR) and cloned into pCR2.1 using the TOPO Cloning Kit (Invitrogen, Carlsbad, CA, USA). The cDNAs were cloned into the spUC vector (Kurihara et al. 2008), which contained the LAT52 promoter, and spUC-LAT52p-FP was cloned into the binary vector pMDC99 (Curtis and Grossniklaus 2003). In RPS5Ap::tdTomato-LTI6b, the RPS5A promoter (Weijers et al. 2001) and tdTomato fused to the start codon of LTI6b (At3g05890) with the (SGGGG)2 linker, which was then cloned into the binary vector pMDC123 (Curtis and Grossniklaus 2003). Transformation of Arabidopsis plants was conducted using the simplified Agrobacterium-mediated transformation method (Narusaka et al. 2010). Then, homozygous and heterozygous transgenic plants were selected, and lines showing strong FP expression were analyzed.

Imaging system

For single- and two-photon imaging, we used a laser-scanning inverted microscope (A1R MP; Nikon, Tokyo, Japan) equipped with a ×25 water immersion objective lens (CFI 75 Apo 25×W MP, WD = 2 mm, NA = 1.10). Two dichroic mirrors, DM495 and DM560, and three emission filters, FF01-479/40, FF02-534/30, and FF01-578/105, were used (Semrock, Rochester, NY, USA). For single-photon excitation, using 488-nm Ar and 561-nm diode lasers, the signal was detected with a photomultiplier tube (PMT) detector. For multiphoton excitation, we used a Ti: sapphire femtosecond oscillator pumped by a pulse laser and a 80-MHz repetition rate Ti:sapphire femtosecond regenerative amplifier (Mai Tai DeepSee 690–1040 nm; Spectra-Physics, Mountain View, CA, USA). In our system, although the Ti:sapphire laser provides 350 nm in the useable tuning range (690–1040 nm, Mai Tai DeepSee; Spectra-Physics), a microscopic system is available for up to 1000 nm. Emitted fluorescence signals were detected using Non-Descanned GaAsP PMT Detectors (Nikon). Laser power delivered to samples and detectors settings were adjusted to avoid saturated pixels and reduce background noise for each experiment. Input and output laser power at each wavelength in this study is shown in Fig. S1. Each image was processed with NIS-Elements AR 4.10 software (Nikon) to create maximum-intensity projection images. Adobe Photoshop CS4 (Adobe Systems, Inc., San Jose, CA, USA) and ImageJ ( were used to optimize the images.

Spectrum imaging of mature pollen grains and root tips

Newly opened flowers were taken from LAT52p::FPs, which expresses mTFP1, sGFP, Venus, TagRFP, and mRFP. Pollen grains were spread on the surface of a coverslip, and a mixture of pollen grains expressing the FPs was excited at 800-1000 nm. Roots of 2-week-old seedlings from WT and RPS5Ap::tdTomato-LTI6b were also observed. The infrared laser power was adjusted as follows to compare images: 800 nm (12.0 %), 850 nm (10.0 %), 900 nm (10.0 %), 920 nm (15.0), 950 nm (20.0 %), 980 nm (25.0 %), and 1000 nm (50.0 %). Input and output laser power at each wavelength is shown in Fig. S1. Emitted fluorescence signals were detected using a 32-channel PMT array detector ranging from 463.9 to 649.2 nm at 6.0-nm intervals. Spectrum analysis and adding color were also processed by NIS-Elements (Nikon).

Deep imaging inside an intact root and pistil

Deep imaging was performed using the same microscope system described above. Roots of 2-week-old seedlings from RPS5Ap::H2B-sGFP and RPS5Ap::tdTomato-LTI6b were placed on a coverslip with water. Unpollinated and pollinated (1 day after pollination (1DAP)) pistils from the FGR8.0 plant were placed on a coverslip with culture media (Mizuta et al. 2014). Emitted fluorescence signals were detected using the Non-Descanned GaAsP PMT detector (Nikon). Z-stack images were taken for projection images and optical sections. Images were processed with the NIS-Elements (Nikon) and ImageJ to create maximum-intensity projection images.


Near-infrared two-photon excitation reduced chloroplast autofluorescence

In plants, light photons are mainly absorbed by photosynthetic pigments such as chlorophyll located in the chloroplasts (Tirlapur and König 2001b; Müller et al. 2013). In Arabidopsis, two chlorophyll-related peaks, one minor at 610 nm from chlorophyll a and one major at 680 nm from chlorophyll b, were observed by single-photon excitation (Langhans and Meckel 2014). To analyze the chlorophyll autofluorescence in Arabidopsis with 2PEM, petioles expressing the GFP-labeled actin filament (GFP-mTalin; Oikawa et al. 2003; Kadota et al. 2009) were studied. Petiole cells are extended longitudinally, which facilitates the observation of cellular components. Figure 1 compares chlorophyll autofluorescence under confocal (single-photon, left) and multiphoton (two-photon, right) excitation. At emissions between 500 and 550 nm, the image quality and GFP signal intensity were similarly intermediate to those from single-photon excitation at 488 nm (Fig. 1, upper left) and two-photon excitation at 1000 nm (Fig. 1, upper right). In contrast, at 570–620 nm, the signal intensity of chlorophyll autofluorescence in the confocal image (Fig. 1, middle left) was much brighter than that from two-photon excitation (Fig. 1, middle right). Under single-photon excitation, detection of red-emitting FPs such as mRFP (emission peak is 607 nm) could be interfered by chlorophyll autofluorescence, which obstructs the emission of such FPs (Chapman et al. 2005). This result demonstrated that two-photon excitation allows imaging of red FPs in plant tissues by eliminating chlorophyll autofluorescence.
Fig. 1

Comparison of single- and two-photon imaging of mouse actin filaments in the petiole of GFP-mTalin. Petioles were observed under both single-photon (488 nm) and two-photon (1000 nm) excitation. Images were collected using FF01-534/30 (500–550 nm, green) and FF01-578/105 (570–620 nm, red) filters. To compare the images at each excitation wavelength, the intensity values were adjusted to the maximum intensity. Z-stack images were taken using 32 z-planes with 3-μm intervals for maximum-intensity projection. Scale bars = 100 μm

Small incremental changes in the excitation wavelength significantly affect the emission intensities of FPs in two-photon excitation (Salomonnson et al. 2012). We analyzed the relationship between the two-photon excitation wavelength and plant autofluorescence in 2PEM. To compare the two-photon images at different excitation wavelengths, the leaf of GFP-mTalin was observed at near-infrared, 800, 900, and 1000 nm (Fig. 2). For excitation at 800 nm, the GFP signals were not clear at emissions in the range of 500–550 nm, whereas chloroplasts were strongly excited and emitted in the 570–620 nm range (Fig. 2, left). Autofluorescence of chloroplasts was evident not only in the 500–550 nm range, but also in the 570–620 nm range. In contrast, in response to excitation at 900 nm, the chloroplast autofluorescence was weaker than 800 nm in the 500–550 nm and 570–620 nm ranges (Fig. 2, middle). The filamentous structures of GFP-fused cytoplasmic cables and those around the chloroplast peripheral region (Kadota et al. 2009) were observed at a range of 500–550 nm emissions (Fig. 2, arrowheads). Furthermore, two-photon excitation at 1000 nm significantly diminished autofluorescence at a range of 500–550 nm emissions (Fig. 2, right). Although the GFP signal at 1000 nm is lower than at 900 nm, autofluorescence in the range of 570–620 nm was also less than at other excitation wavelengths (Fig. 2, right). These results suggested that the longer wavelength excitation by 2PEM is useful to observe FPs in Arabidopsis tissues by reducing chloroplast autofluorescence.
Fig. 2

Comparisons among excitation wavelengths of two-photon imaging of the leaf surface of GFP-mTalin. Optical sections of a leaf were imaged by two-photon microscopy at 800, 900, and 1000 nm excitation. To compare the images at each excitation wavelength, the infrared laser power was adjusted to 17.4 in NIS-Elements. The following laser power was used in excitation at each wavelength: 800 nm (8.4 %), 900 nm (12.5 %), and 1000 nm (40.0 %). Input and output laser power at each wavelength is shown in Fig. S1. Images were collected with FF01-534/30 (500–550 nm, green) and FF01-578/105 (570–620 nm, red) filters. Z-stack images were taken using 12 z-planes with 10-μm intervals for maximum-intensity projection. Arrowheads indicate the filamentous structures of GFP-fused cytoplasmic cables and those around the chloroplast peripheral region. Scale bars = 100 μm

The near-infrared wavelength, 1000 nm, reduced autofluorescence compounds in both pollen and roots

The chloroplast and other endogenous plant fluorophores, such as cutin, suberin, sporopollenin, and exine, disturb the targeted fluorescent signals (Chapman et al. 2005; Müller et al. 2013). In Arabidopsis, chloroplast development is suppressed in roots and pollen, and mature pollen grains from LAT52p::TagRFP heterozygous plants and root tips of RPS5Ap::tdTomato-LTI6b homozygous plants were observed using 2PEM (Fig. 3). Under 1000 nm excitation, TagRFP proteins in pollen were excited (Fig. 3a, arrow), whereas WT pollen was not (Fig. 3a, arrowhead). In contrast, for both 800 and 900 nm excitation, the TagRFP and WT pollen grains showed green fluorescence (500–550 nm, Fig. 3). Due to the absence of chlorophyll, this green autofluorescence, especially strong at 800 nm excitation, came from cell walls or other cellular components. Similar green autofluorescence was observed in roots at shorter excitation wavelengths (Fig. 3b). Figure 3b, c shows the WT and tdTomato-expressing root tip at 800, 900, and 1000 nm excitation, respectively. In the root tip of the transgenic plant, the tdTomato signal was clearly detected in the plasma membrane at 1000 nm excitation. In contrast, the tdTomato signal was not sufficient at 800 and 900 nm, and green autofluorescence was evident, which was derived from at least two compounds in the cell wall (Fig. 3b, arrow) and cell contents (Fig. 3b, arrowhead) in the root tip.
Fig. 3

Comparisons among the two-photon excitation wavelengths at 800, 900, and 1000 nm on pollen grains and roots. Spectral images and individual channel images were shown. a Two-photon image of pollen grains from a LAT52p::TagRFP heterozygous plant. Z-stack images were taken using 3 z-planes with 5-μm intervals for maximum projection intensity. The arrow and arrowhead indicate TagRFP-expressing and wild-type pollen grains, respectively. To compare the images between excitation wavelengths, the infrared laser power was adjusted to 15 in NIS-Elements. b, c Two-photon image of a root tip from WT (b) and RPS5Ap::tdTomato-LTI6b (c). Z-stack images were taken using 7 z-planes with 15-μm intervals for maximum projection intensity. The arrow and arrowhead indicate different autofluorescence from the root cell wall and columella region, respectively. Images were acquired in sequential bandwidths of 6-nm spanning the wavelength range of 463.9–649.2 nm to generate a lambda stack containing 32 images. Scale bars = 100 μm

For clear two-photon imaging of FPs without autofluorescence, the autofluorescence spectrum was further examined. Figure 4 shows the two-photon emission spectra of the mature pollen grains from LAT52p::FP homozygous transgenic plants. Each mature pollen grain expressing mTFP1, sGFP, Venus, TagRFP, and mRFP was observed under two-photon excitation at 800, 900, 980, and 1000 nm (Fig. 4). Excitation spectra of all examined pollen grains showed a single, narrow peak at both 980 and 1000 nm, while large differences in the absorption spectra were evident at shorter excitation wavelengths. Autofluorescence in the ranges of 450–500 and 480–550 nm was emitted under two-photon excitation at 800 and 900 nm, respectively (Fig. 4). These blue to yellow emissions appeared to be autofluorescence from pollen cell walls and other cellular components. At the main peak of these compounds, autofluorescence obstructs the emission of blue to yellow-emitting FPs, such as mTFP1 (Ex. 492 nm), sGFP (Ex. 512 nm), and Venus (Ex. 528 nm). Note that the fluorescence emission spectra were separated by a narrow peak in mTFP1, sGFP, and Venus, although emissions from autofluorescence were also added to these absorption spectra at excitation wavelengths of 800 and 900 nm.
Fig. 4

Comparisons of the two-photon excitation fluorescence spectra of the mature pollen from LAT52p::FP homozygous plants. The emission spectrum of each pollen grain is shown as absolute (a) and normalized (b) fluorescence intensities. Mature pollen grains from LAT52p::FP were spread on the cover glass and observed under two-photon excitation at 800-1000 nm. The x-axis is the spectrum profile of each pollen grain expressing fluorescent proteins, and the y-axis is the absolute (a) and normalized (b) mean value of the channel inside the regions of interest in pollen. The fluorescent intensity was normalized with each peak of fluorescence spectra normalized as 1.0

From these results, shorter excitation wavelengths (e.g., 800 and 900 nm) were not better for observing FPs in Arabidopsis tissues. For clear observation without green plant autofluorescence, a longer excitation wavelength (e.g., 1000 nm) was optimal (Fig. 2). Choosing both suitable FPs and excitation wavelengths is important, giving consideration to the targeted tissues and their developmental stages.

Simultaneous two-photon excitation of FPs for multicolor imaging

Two-photon absorption spectra are often broader than their single-photon equivalents (Benninger and Piston 2013); thus, the femtosecond pulse laser allows simultaneous excitation of multiple fluorophores by a single wavelength (Xu et al. 1996; Drobizhev et al. 2011). This feature allowed reducing photodamage for specimens and achieves simultaneous multicolor imaging. To analyze the proper wavelength for simultaneous excitation, we used multicolor 2PEM on mature pollen grains expressing five different FPs (Figs. S2 and 5). The mixture of pollen grains on a coverslip was simultaneously subjected to two-photon excitation at each wavelength (Fig. 5a). The pollen grains expressing mTFP1 were strongly excited from 800 to 950 nm, whereas they were much less excited at 1000 nm (Fig. 5b). The pollen grains expressing sGFP and Venus were relatively highly excited at all excitation wavelengths, although both were less excited at 800 nm (Fig. 5b). The pollen grains expressing TagRFP and mRFP were excited at 800 and above 980 nm and much less excited from 850 to 900 nm (Fig. 5b). At these eight wavelengths, pollen grains expressing five FPs were simultaneously excited at both 800 and 980 nm. When the excitation laser is tuned to 780 nm, it should simultaneously excite the mKalama1 (blue) and TagRFP (orange) FPs in HEK293 cells (Tillo et al. 2010). However, a longer wavelength is known to be better for deep imaging to avoid photodamage (Kawakami et al. 2013), and two-photon excitation at 800 nm caused cellular autofluorescence (Figs. 2, 3, and 4). Thus, a two-photon excitation wavelength at 980 nm is better for simultaneous excitation of FPs and multicolor imaging.
Fig. 5

Multicolor two-photon imaging of pollen grains expressing FP, mTFP1 (TF), sGFP (sG), Venus (V), TagRFP (TR), and mRFP (mR). a Large image of a mixture of pollen grains on a coverslip under two-photon excitation at 800 and 1000 nm. Large images at other excitation wavelengths are shown in Fig. S2. b Each pollen grain under the two-photon excitation at 800-1000 nm. To compare the images at each excitation wavelength, the infrared laser power was adjusted to 23.7 in NIS-Elements. The following laser power was used in excitation at each wavelength: 800 nm (12.0 %), 850 nm (10.0 %), 900 nm (10.0 %), 920 nm (15.0 %), 950 nm (20.0 %), 980 nm (25.0 %), and 1000 nm (50.0 %). Images were acquired in sequential bandwidths of 6 nm spanning the wavelength range of 463.9–649.2 nm to generate a lambda stack containing 32 images. Scale bars = 30 μm (a) and 100 μm (b)

High-resolution imaging inside roots and pistils

Our results showed that 2PEM, with longer wavelength excitation (e.g., 1000 nm), reduces plant autofluorescence. Additionally, longer wavelength excitation (e.g., 980 nm) enabled us to perform multicolor two-photon imaging. Thus, we performed deep imaging of intact root tips and pistils by 2PEM at 980–1000 nm. To evaluate the spatial resolution of root tips in the z-axial direction, we performed Z-stack image acquisition and reconstructed X–Y and X–Z sectioning images for both single- and two-photon excitation (Fig. 6a–d). Figure 6a, b shows maximum-intensity images in the center of a root tip of RPS5Ap::H2B-sGFP. In the two-photon excitation image, the X–Y image contrast at the center of the root tip was higher than that from single-photon excitation. In single-photon excitation, the reconstructed X–Z image was blurred, but these images were sharp in two-photon excitation. Signal intensity decreased at the center of the root tip in both the elongation and transition zones.
Fig. 6

Comparisons of imaging penetration in the X–Y and X–Z planes for single- and two-photon excitation of a root tip. a, b Optical sections generated from single- and two-photon excitation fluorescence images of RPS5Ap::H2B-sGFP collected at intervals of 1-μm steps with 137 planes from the side surface of a root tip. c, d Optical sections generated from single- and two-photon excitation fluorescence images of RPS5Ap::tdTomato-LTI6b collected at intervals of 1-μm steps with 150 planes from the side surface of a root tip. X–Z plane images at the center of a root were reconstructed from the Z-stack image shown to the left. Each image was cross-sectioned along the colored lines shown to the right (1 elongation zone, 2 transition zone, 3 meristematic zone). The detection objective lens shows the upper side of the X–Z plane images. Each image was normalized to the maximum signal intensity. Scale bars = 50 μm

The nucleus of cells in the meristematic zone was clear in two-photon images (Fig. 6b), and similar results were obtained from the root tip of RPS5Ap::tdTomato-LTI6b (Fig. 6c, d). In the two-photon excitation image, the X–Y image contrast at the center of the root tip was higher than that from single-photon excitation, and the cell shape was clearer. In the reconstructed X–Z images, cell shapes at the center of both transition and meristematic zones were clear in the two-photon image (Fig. 6d). Although axial resolution decreased as depth increased, two-photon excitation provided higher resolution than single-photon excitation. These results demonstrated the advantage of 2PEM for internal imaging of intact roots.

We attempted multicolor deep imaging of intact pistils using 2PEM at 980 and 990 nm. We compared the results from the deep imaging of pistils using single- and two-photon excitation. Figure 7 shows the inside of the unpollinated pistil of FGR8.0. In this transformant, the expression was shown of a reporter that combined marker genes for synergid cells (DD2::NLS_3x GFP), egg cells (EC1::NLS_3x dsRed), and central cells (DD22::YFP) (Völz et al. 2013). The two nuclei in each synergid cell and the nuclei in egg cells were clearly identified in the isolated ovules (Völz et al. 2013). Z-stack images from the ovary surface to 60–130-μm depths were taken using 15 z-planes with 5-μm intervals for maximum intensity. Using single-photon excitation, the nuclei in the synergid and egg cells were unclear (Fig. 7a, left). Using two-photon excitation, the synergid and egg cell nuclei could be clearly identified inside an intact pistil at a depth of 60–130 μm (Fig. 7a, right). When the ovules were in a proper orientation, the two synergid nuclei were clearly identified at a depth of 100–150 μm (magnified image in Fig. 7a). Light scattering and autofluorescence from the ovary wall also decreased under two-photon excitation (Fig. 7awhite arrows). Deeper tissues, such as the nucleus in the deeper ovules, were also clearly visualized observed in a two-photon image (Fig. 7a, yellow arrows).
Fig. 7

Comparisons of imaging penetration depth between single- and two-photon excitation. a Optical sections of unpollinated pistil from FGR8.0 were imaged by single-photon excitation at 488 and 561 nm and two-photon excitation at 980 nm (laser power, 20 %). Z-stack images at 60–130-μm depth from the ovary surface were taken using 15 z-planes with 5-μm intervals. DD2::NLS_3x GFP (green) and EC1::NLS_3x dsRed (magenta) are shown in color. SY synergid nucleus, EC egg cell nucleus. b Optical sections of a 1 day after pollination (1DAP) pistil from FGR8.0. Two-photon images were taken with two-photon excitation at 990 nm (laser power, 20 %). Z-stack images at 65–130-μm depth from the ovary surface were taken using 14 z-planes with 5-μm intervals. Maximum-intensity projections of a fluorescence image stack were obtained with NIS-Elements software. a Nucleus in the apical cell of a one-cell-stage embryo; b nucleus in the basal cell of a one-cell-stage embryo. White and yellow arrows indicate the autofluorescence from an ovary wall and deeper ovules. Scale bars = 100 μm (upper panels) and 20 μm (lower panels)

Then, 1DAP, the pistil from an FGR8.0 plant was also observed by 2PEM. Autofluorescence from the ovary cell wall was stronger than that for an unpollinated pistil (Fig. 7a) due to silique development. Thus, 990 nm was used for two-photon excitation. In the intact silique, each nucleus in the synergid and egg cells was clearly identified (Fig. 7b). Unexpectedly, a one-cell embryo was evident in some ovules (magnified image in Fig. 7b). These results indicated that 2PEM enables clear discrimination of each nucleus in ovules during fertilization, even deep inside the intact pistil.


A biological sample is composed of heterogeneous tissues in spatial variation, which causes scattering of excitation light, with longer wavelengths being scattered less than shorter wavelengths (Benninger and Piston 2013). In this study, we found that 2PEM of orange FPs at 1000 nm excitation allowed us to clearly see the cellular components in deep plant tissues by eliminating autofluorescence. In plant studies, GFP is generally used for in vivo imaging of cells and tissues to avoid chlorophyll autofluorescence, but some red FPs, such as mRFP, are not appropriate for in vivo imaging in plant tissues. Berg (2004) and Berg and Beachy (2008) reported emission spectra of Arabidopsis autofluorescent compounds such as sporopollenin, cutin, suberin, lignin, and chlorophyll using two-photon microscopy. Most of these emissions were in the blue to green region (∼440 to ∼540 nm), potentially overlapping with cyan, green, and yellow FPs, which suggested that emissions from autofluorescence were also added to these fluorescent spectra. In contrast, some red FPs are known to show lower quantum yield. Especially in two-photon excitation, some red FPs (e.g., mCherry or mStrawberry) show extremely lower quantum yields than single-photon excitation (Drobizhev et al. 2011). Thus, one must identify the FP that is best for 2PEM in plant cells. Orange FPs were useful for clear two-photon imaging in plant tissues, as TagRFP had a higher value than other orange–red FPs under the tuning range (700–1000 nm) of a Ti:sapphire laser (Drobizhev et al. 2009). TagRFP matures quickly and can be expressed in many different types of organisms; it also works well as a fusion protein (Merzlyak et al. 2007). Drobizhev et al. (2009) suggested that tdTomato has the largest value at 1000–1100 nm excitation, which is a three- to fourfold improvement in two-photon brightness compared to enhanced GFPs. Taking these findings into account, orange FPs such as TagRFP and tdTomato seem to be the best for two-photon imaging in Arabidopsis in eliminating plant autofluorescence. Orange fluorophores, with two-photon excitation and longer wavelengths, will enable us to achieve greater imaging depth in plant tissues.

In recent decades, longer wavelengths (i.e., >1000 nm) have been used in two-photon microscopy. The imaging depth at 1280 nm excitation was deeper than at a lower wavelength (Kobat et al. 2009), and lowest light attenuation occurred at 1200–1300 nm excitation (Chen et al. 2002; Tsai et al. 2006). Some reports showed an improved imaging depth using infrared lasers. The far-red FP was excited with a Ti:sapphire laser at 1120–1230 nm excitation from an optical parametric oscillator (Tsai et al. 2006) and a Cr:forsterite laser at −1300 nm excitation (Kobat et al. 2009). Recent reports demonstrated high resolution of subcortical structures within an intact mouse brain using noninvasive three-photon fluorescence microscopy at 1700 nm excitation (Horton et al. 2013). Another study demonstrated that two-photon excitation digital-scanned light-sheet microscopy (2p-DSLM) achieves high-speed imaging and deep penetration in fruit fly and zebrafish (Truong et al. 2011). The higher-order nonlinear excitation with longer excitation wavelengths will facilitate biological investigations at greater depths in plant tissues.

Longer wavelength excitation also provides advantages for simultaneous imaging. FPs having different spectral characteristics are useful for simultaneous imaging of multiple proteins as intracellular biosensors (Verkhusha and Lukyanov 2004). However, the combination of FPs that enables simultaneous imaging is limited. Some improved FPs with a large Stokes shift have been reported (Kogure et al. 2008). These FPs allow us to do multicolor imaging by simultaneous two-photon excitation. Several FPs have an interesting feature in that the two-photon excitation peaks are blueshifted with respect to the single-photon absorption peaks at twice the wavelength (Albota et al. 1998; Drobizhev et al. 2011). This blueshift feature is remarkable in some FPs such as DsRed2 and enhanced GFP (Drobizhev et al. 2011).

Both high quantum yield and a high extinction coefficient allow the use of lower levels of excitation light, which is desirable in reducing specimen damage due to phototoxicity. Recently, multicolor- and four-dimensional imagings were successful when using the narrow range of a band-pass filter to reduce background signals (Kitano and Okada 2012). 2PEM with a dual-laser beam system was also reported (Zinselmeyer et al. 2009). The wavelength scans with various FPs and the use of filters may suffice in finding a good absorption band in the absence of plant autofluorescence. The same types of tissues may vary in autofluorescence, depending on the condition of the plant tissues (Roshchina 2012). Selecting proper FPs with the best excitation wavelength is important, while considering autofluorescence for multicolor deep imaging by 2PEM in living plant tissues.

Two-photon excitation penetrates deeper into the specimen than single-photon microscopy. For single-photon excitation, observing deep inside plant organs is difficult because of scattering structures (multiple cell layers) and components (Fig. 6a, c). In contrast, the signal intensity is retained under the two-photon excitation (Fig. 6b, d). Although the signal intensity of the single-photon images decreases with depth, the depth of imaging penetration from 2PEM was approximately twice that of single-photon imaging (Fig. 6). 2PEM has become a powerful tool for deep imaging of plant’s living tissues. In Arabidopsis, 2PEM facilitated studying the behavior of sperm cells at three steps in an ovule during double fertilization (Hamamura et al. 2011, 2012). Kurihara et al. (2013) isolated an Arabidopsis ovule by 2PEM that allowed clear discrimination of each nucleus in embryo cells at the globular stage. 2PEM is also useful for noninvasive imaging, as it reduces cell damage and enables the imaging of live cells. Cheung et al. (2010) attempted to observe the in vivo PT growth in intact pistils using 2PEM and observed the GFP-labeled PTs deep inside the pollinated pistils. After observations, the observed plant could be further cultured in the greenhouse and seed set was confirmed (Cheung et al. 2010). In the future, studies focusing on individual plant organs by sequential imaging using 2PEM will be able to do, such as from before fertilization to seed development on intact plants. Further advances of live-cell analysis in vivo by 2PEM will facilitate more in-depth analyses in the plant biological sciences.



We thank Dr. A. Kadota (Tokyo Metropolitan University) and Dr. Y. Sato (Nagoya University) for kindly providing seeds of GFP-mTalin and Dr. R. Groß-Hardt (University of Bremen) for kindly providing seeds of FGR8.0. We thank Dr. D. Maruyama (Nagoya University) and Dr. Y. Hamamura (Université de Montréal) for providing plasmids. We thank S. Nasu, T. Nishii and T. Shinagawa for plant culture and S. Nagahara for providing plant material. This work was supported by grants from the Japan Science and Technology Agency (ERATO project to T.H.) and the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 26840104 to Y.M.).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2014_754_MOESM1_ESM.tif (160 kb)
Fig. S1Input and output laser power at each excitation wavelengths. The input laser power on the NIS-Elements software is shown as a percentage. Each output laser power on the NIS-Elements software was measured by internal laser sensor in microscopy. (TIFF 159 kb)
709_2014_754_Fig8_ESM.gif (82 kb)

High resolution image (GIF 82 kb)

709_2014_754_MOESM2_ESM.tif (851 kb)
Fig. S2Large images of a mixture of pollen grains on a coverslip under two-photon excitation at 850, 900, 920, 950, and 980 nm. Each pollen grain expressing following the fluorescent proteins was mixed: mTFP1 (TF), sGFP (sG), Venus (V), TagRFP (TR), and mRFP (mR). To compare the images among each excitation wavelength, the infrared laser power was adjusted to 23.7 in NIS-Elements. The following laser power was used at each excitation wavelength: 850 nm (10.0 %), 900 nm (10.0 %), 920 nm (15.0 %), 950 nm (20.0 %), and 980 nm (25.0 %). Images were acquired in sequential bandwidths of six nanometers spanning the wavelength range of 463.9–649.2 nm to generate a lambda stack containing 32 images. Scale bar = 100 μm (TIFF 851 kb)
709_2014_754_Fig9_ESM.gif (106 kb)

High resolution image (GIF 105 kb)


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Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Yoko Mizuta
    • 1
    • 2
  • Daisuke Kurihara
    • 1
    • 2
  • Tetsuya Higashiyama
    • 1
    • 2
    • 3
  1. 1.Graduate School of ScienceNagoya UniversityNagoyaJapan
  2. 2.JST, ERATO, Higashiyama Live-Holonics ProjectNagoya UniversityNagoyaJapan
  3. 3.Institute of Transformative Bio-Molecules (WPI-ITbM)Nagoya UniversityNagoyaJapan

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