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

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Abstract

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.

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References

  • Adachi S, Minamisawa K, Okushima Y, Inagaki S, Yoshiyama K, Kondou Y, Kaminuma E, Kawashima M, Toyoda T, Matsui M, Kurihara D, Matsunaga S, Umeda M (2011) Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis. Proc Natl Acad Sci U S A 108:10004–10009

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Albota MA, Xu C, Webb WW (1998) Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm. Appl Opt 37:7352–7356

    Article  CAS  PubMed  Google Scholar 

  • Benninger RK, Piston DW (2013) Two-photon excitation microscopy for the study of living cells and tissues. Curr Protoc Cell Biol Chapter 4:Unit 4.11.1–24

  • Berg RH (2004) Evaluation of spectral imaging for plant cell analysis. J Microsc 214:174–181

    Article  CAS  PubMed  Google Scholar 

  • Berg RH, Beachy RN (2008) Fluorescent protein applications in plants. Methods Cell Biol 85:153–177

    Article  CAS  PubMed  Google Scholar 

  • Bousso P, Robey EA (2004) Dynamic behavior of T cells and thymocytes in lymphoid organs as revealed by two-photon microscopy. Immunity 21:349–355

    Article  CAS  PubMed  Google Scholar 

  • Brakenhoff GJ, van der Voort HT, van Spronsen EA, Linnemans WA, Nanninga N (1985) Three-dimensional chromatin distribution in neuroblastoma nuclei shown by confocal scanning laser microscopy. Nature 317:748–749

    Article  CAS  PubMed  Google Scholar 

  • Cahalan MD, Parker I, Wei SH, Miller MJ (2003) Real-time imaging of lymphocytes in vivo. Curr Opin Immunol 15:372–377

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Centonze VE, White JG (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75:2015–2024

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chapman S, Oparka KJ, Roberts AG (2005) New tools for in vivo fluorescence tagging. Curr Opin Plant Biol 8:565–573

    Article  CAS  PubMed  Google Scholar 

  • Chen IH, Chu SW, Sun CK, Cheng PC, Lin BL (2002) Wavelength dependent damage in biological multi-photon confocal microscopy: a micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources. Opt Quant Electron 34:1251–1266

    Article  Google Scholar 

  • Cheung AY, Boavida LC, Aggarwal M, Wu HM, Feijó JA (2010) The pollen tube journey in the pistil and imaging the in vivo process by two-photon microscopy. J Exp Bot 61:1907–1915

    Article  CAS  PubMed  Google Scholar 

  • Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Denk W, Svoboda K (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18:351–357

    Article  CAS  PubMed  Google Scholar 

  • Drobizhev M, Tillo S, Makarov NS, Hughes TE, Rebane A (2009) Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. J Phys Chem B 113:855–859

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Drobizhev M, Makarov NS, Tillo SE, Hughes TE, Rebane A (2011) Two-photon absorption properties of fluorescent proteins. Nat Methods 8:393–399

    Article  CAS  PubMed  Google Scholar 

  • Feijó JA, Moreno N (2004) Imaging plant cells by two-photon excitation. Protoplasma 223:1–32

    Article  PubMed  Google Scholar 

  • Hamamura Y, Saito C, Awai C, Kurihara D, Miyawaki A, Nakagawa T, Kanaoka MM, Sasaki N, Nakano A, Berger F, Higashiyama T (2011) Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana. Curr Biol 21:497–502

    Article  CAS  PubMed  Google Scholar 

  • Hamamura Y, Nagahara S, Higashiyama T (2012) Double fertilization on the move. Curr Opin Plant Biol 15:70–77

    Article  PubMed  Google Scholar 

  • Helmchen F, Denk W (2002) New developments in multiphoton microscopy. Curr Opin Neurobiol 12:593–601

    Article  CAS  PubMed  Google Scholar 

  • Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940

    Article  CAS  PubMed  Google Scholar 

  • Horton NG, Wang K, Kobat D, Clark CG, Wise FW, Schaffer CB, Xu C (2013) In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat Photonics 7. doi:10.1038/nphoton.2012.336

  • Kadota A, Yamada N, Suetsugu N, Hirose M, Saito C, Shoda K, Ichikawa S, Kagawa T, Nakano A, Wada M (2009) Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis. Proc Natl Acad Sci U S A 106:13106–13111

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kawakami R, Sawada K, Sato A, Hibi T, Kozawa Y, Sato S, Yokoyama H, Nemoto T (2013) Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser. Sci Rep 3:1014

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kitano M, Okada T (2012) Four-dimensional tracking of lymphocyte migration and interactions in lymph nodes by two-photon microscopy. Methods Enzymol 506:437–454

    Article  CAS  PubMed  Google Scholar 

  • Kobat D, Durst ME, Nishimura N, Wong AW, Schaffer CB, Xu C (2009) Deep tissue multiphoton microscopy using longer wavelength excitation. Opt Express 17:13354–13364

    Article  PubMed  Google Scholar 

  • Kogure T, Kawano H, Abe Y, Miyawaki A (2008) Fluorescence imaging using a fluorescent protein with a large Stokes shift. Methods 45:223–226

    Article  CAS  PubMed  Google Scholar 

  • König K (2000) Multiphoton microscopy in life sciences. J Microsc 200:83–104

    Article  PubMed  Google Scholar 

  • Kurihara D, Matsunaga S, Uchiyama S, Fukui K (2008) Live cell imaging reveals plant aurora kinase has dual roles during mitosis. Plant Cell Physiol 49:1256–1261

    Article  CAS  PubMed  Google Scholar 

  • Kurihara D, Hamamura Y, Higashiyama T (2013) Live-cell analysis of plant reproduction: live-cell imaging, optical manipulation, and advanced microscopy technologies. Dev Growth Differ 55:462–473

    Article  CAS  PubMed  Google Scholar 

  • Laiho LH, Pelet S, Hancewicz TM, Kaplan PD, So PT (2005) Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra. J Biomed Opt 10:024016

    Article  PubMed  Google Scholar 

  • Langhans M, Meckel T (2014) Single-molecule detection and tracking in plants. Protoplasma 251:277–291

    Article  CAS  PubMed  Google Scholar 

  • Maruyama D, Hamamura Y, Takeuchi H, Susaki D, Nishimaki M, Kurihara D, Kasahara RD, Higashiyama T (2013) Independent control by each female gamete prevents the attraction of multiple pollen tubes. Dev Cell 25:317–323

    Article  CAS  PubMed  Google Scholar 

  • Merzlyak EM, Goedhart J, Shcherbo D, Bulina ME, Shcheglov AS, Fradkov AF, Gaintzeva A, Lukyanov KA, Lukyanov S, Gadella TW, Chudakov DM (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4:555–557

    Article  CAS  PubMed  Google Scholar 

  • Mizuta Y, Kurihara D, Higashiyama T (2014) Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes. Plant J 78:516–526

    Article  CAS  PubMed  Google Scholar 

  • Molitoris BA, Sandoval RM (2005) Intravital multiphoton microscopy of dynamic renal processes. Am J Physiol Renal Physiol 288:F1084–F1089

    Article  CAS  PubMed  Google Scholar 

  • Müller SM, Galliardt H, Schneider J, Barisas BG, Seidel T (2013) Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells. Front Plant Sci 4:413

    PubMed Central  PubMed  Google Scholar 

  • Narusaka M, Shiraishi T, Iwabuchi M, Narusaka Y (2010) The floral inoculating protocol: a simplified Arabidopsis thaliana transformation method modified from floral dipping. Plant Biotechnol 27:349–351

    Article  Google Scholar 

  • Oikawa K, Kasahara M, Kiyosue T, Kagawa T, Suetsugu N, Takahashi F, Kanegae T, Niwa Y, Kadota A, Wada M (2003) Chloroplast unusual positioning1 is essential for proper chloroplast positioning. Plant Cell 15:2805–2815

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Pawley JB (2006) Handbook of biological confocal microscopy, 3rd edn. Springer, New York

    Book  Google Scholar 

  • Roshchina VV (2012) Vital autofluorescence: application to the study of plant living cells. Int J Spectrosc 124672

  • Rost FWD (1995) Fluorescence microscopy. Cambridge University Press, Cambridge

    Google Scholar 

  • Rubart M (2004) Two-photon microscopy of cells and tissue. Circ Res 95:1154–1166

    Article  CAS  PubMed  Google Scholar 

  • Salomonnson E, Mihalko LA, Verkhusha VV, Luker KE, Luker GD (2012) Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy. J Biomed Opt 17:96001

    Article  PubMed  Google Scholar 

  • Schwille P, Haupts U, Maiti S, Webb WW (1999) Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys J 77:2251–2265

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239

    Article  CAS  PubMed  Google Scholar 

  • So PT, Dong CY, Masters BR, Berland KM (2000) Two-photon excitation fluorescence microscopy. Annu Rev Biomed Eng 2:399–429

    Article  CAS  PubMed  Google Scholar 

  • Tillo SE, Hughes TE, Makarov NS, Rebane A, Drobizhev M (2010) A new approach to dual-color two-photon microscopy with fluorescent proteins. BMC Biotechnol 10:6

    Article  PubMed Central  PubMed  Google Scholar 

  • Tirlapur UK, König K (2001a) Femtosecond near-infrared laser pulse induced strand breaks in mammalian cells. Cell Mol Biol (Noisy-le-grand) 47:OL131–OL134

    CAS  Google Scholar 

  • Tirlapur UK, König K (2001b) Femtosecond near-infrared lasers as a novel tool for non-invasive real-time high-resolution time-lapse imaging of chloroplast division in living bundle sheath cells of Arabidopsis. Planta 214:1–10

    Article  CAS  PubMed  Google Scholar 

  • Truong TV, Supatto W, Koos DS, Choi JM, Fraser SE (2011) Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat Methods 8:757–760

    Article  CAS  PubMed  Google Scholar 

  • Tsai TH, Lin CY, Tsai HJ, Chen SY, Tai SP, Lin KH, Sun CK (2006) Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section. Opt Lett 31:930–932

    Article  CAS  PubMed  Google Scholar 

  • Twell D, Wing R, Yamaguchi J, McCormick S (1989) Isolation and expression of an anther-specific gene from tomato. Mol Gen Genet MGG 217:240–245

    Article  CAS  PubMed  Google Scholar 

  • Verkhusha VV, Lukyanov KA (2004) The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol 22:289–296

    Article  CAS  PubMed  Google Scholar 

  • Völz R, Heydlauff J, Ripper D, von Lyncker L, Groß-Hardt R (2013) Ethylene signaling is required for synergid degeneration and the establishment of a pollen tube block. Dev Cell 25:310–316

    Article  PubMed  Google Scholar 

  • Weijers D, Franke-van Dijk M, Vencken RJ, Quint A, Hooykaas P, Offringa R (2001) An Arabidopsis Minute-like phenotype caused by a semi-dominant mutation in a RIBOSOMAL PROTEIN S5 gene. Development 128:4289–4299

    CAS  PubMed  Google Scholar 

  • White JG, Amos WB, Fordham M (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105:41–48

    Article  CAS  PubMed  Google Scholar 

  • Xu C, Williams RM, Zipfel W, Webb WW (1996) Multiphoton excitation cross-sections of molecular fluorophores. Bioimaging 4:198–207

    Article  CAS  Google Scholar 

  • Zinselmeyer BH, Dempster J, Wokosin DL, Cannon JJ, Pless R, Parker I, Miller MJ (2009) Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics. Methods Enzymol 461:349–378

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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.

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Authors and Affiliations

  1. Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan

    Yoko Mizuta, Daisuke Kurihara & Tetsuya Higashiyama

  2. JST, ERATO, Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan

    Yoko Mizuta, Daisuke Kurihara & Tetsuya Higashiyama

  3. Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan

    Tetsuya Higashiyama

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  1. Yoko Mizuta
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Correspondence to Yoko Mizuta.

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Handling Editor: Liwen Jiang

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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)

High resolution image (GIF 82 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)

High resolution image (GIF 105 kb)

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Mizuta, Y., Kurihara, D. & Higashiyama, T. Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues. Protoplasma 252, 1231–1240 (2015). https://doi.org/10.1007/s00709-014-0754-5

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