, 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 



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. S1 Input 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. S2 Large 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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