Vol. 49, No. 2, D Truernit et al PS - PI modified pseudo - Schiff propidium iodide ; mps - PI Z μm GUS mrna Yoshida et al. 2014

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1 特集植物の生長調節 Regulation of Plant Growth & Development Vol.49, No.2, , ,2 1,2 1 2 JST ERATO In vivo deep imaging and optical manipulation by two photon excitation microscopy Yoko Mizuta 1,2 and Daisuke Kurihara 1,2 1 Graduate School of Science, Nagoya University 2 Higashiyama Live - Holonics Project, ERATO, JST 要旨 : Cell - to - cell communication plays an important role in development, reproduction, and environmental responses by coordinating the cell activity in multicellular organisms. In plant development, cell - to - cell communication is especially crucial because organogenesis is a postembryonic and continuous process. However, it is difficult to reveal the dynamics of intercellular communications due to the limitation of accessibility within several cell layers in plants. Recently, two - photon excitation microscopy with near - infrared femtosecond pulse laser provides a non - invasive tool for deep imaging. In this review, we discuss recent deep imaging containing the preparation of specimens and microscopic techniques. Moreover, optical manipulation technique with near - infrared ultrafast pulse laser enables us to disrupt the cell and the protein at the single - cell level. Combination of in vivo deep imaging and optical manipulation, which allow spatial and temporal control of cell activity, provides a new view of the cell - to - cell communication in plant field. はじめに in vitro 2 Helmchen and Denk 2005; Benninger and Piston 植物組織の深部イメージング Moreno et al Müller et al ; 2014 The Japanese Society for Chemical Regulation of Plants

Vol. 49, No. 2, 2014 97 3D Truernit et al.

2 μm GUS mrna Yoshida et al. 2014 PI Z 0.1 μm 2.

2 Vol. 49, No. 2, D Truernit et al PS - PI modified pseudo - Schiff propidium iodide ; mps - PI Z μm GUS mrna Yoshida et al PI Z 0.1 μm 2. 深部イメージングにおける 2 光子励起顕微鏡の利点図 A 2 B nm nm GFP μm 図 2 2 A 80 μm GFP YFP DsRed 980 nm B 80 μm tdtomato 950 nm C 30 μm tdtomato 920 nm 100 μm A 30 μm B, C

3 98 Vol. 49, No. 2, μm 2 Helmchen and Denk 2005 ; Benninger and Piston , A B 光子励起顕微鏡による植物の生体深部イメージング 2 Miller et al ; Stosiek et al mm Kawakami et al Hepler and Gunning Feijó and Moreno 2004 ; Colaço et al in vitro Hamamura et al. 2011, Kurihara et al B C 2 2B 2 2C 80 μm 30 μm μm 2 in vivo Cheung et al A 80 μm Cheung et al

4 Vol. 49, No. 2, UV レーザーによる細胞破壊 1 ; Bargmann and Avery 1995 van den Berg et al van den Berg et al Higashiyama et al UV Higashiyama et al ; Okuda et al UV UV UV UV 5. 近赤外超短パルスレーザーによる細胞破壊 UV 4 Tirlapur and König 2002b König et al nm 110 nm 740 nm 400 m Tirlapur and König 2002a

The Japanese Society for Chemical Regulation of Plants (JSCRP) 100 植物の生長調節図 3 HTR8p::HTR8-GFP のめしべから単離した胚珠の 2 光子励起像胚のう内の胚乳核が GFP 緑により可視化されている赤色は自家蛍光 A Z 軸方向に 3 μm おきに 177 μm 深部まで観察した画像を立体構築した像

2, 2014 図 4 パルスレーザーのパルス幅とピーク出力パルスエネルギー面積に相当が同じ場合パルス幅が短いほどピーク出力は高い図 5 近赤外超短パルスレーザーを用いた雌性配偶体細胞の破壊実験シロイヌナズナの卵細胞特異的に Ca2+ センサー YC3.60 Nagai et al.

5 The Japanese Society for Chemical Regulation of Plants (JSCRP) 100 植物の生長調節図 3 HTR8p::HTR8-GFP のめしべから単離した胚珠の 2 光子励起像胚のう内の胚乳核が GFP 緑により可視化されている赤色は自家蛍光 A Z 軸方向に 3 μm おきに 177 μm 深部まで観察した画像を立体構築した像手前側が観察面胚珠の外側から観察しても胚乳核と細胞の輪郭がはっきり観察できるスケールバーは 100 μm B A の図を Z 軸方向から見たもの上部が観察面数字は観察面からの深さを示す胚乳核は 175 μm 深部でもはっきり見ることができ細胞が胚珠内でどのように配置しているかを立体的に把握することができる Vol. 49, No. 2, 2014 図 4 パルスレーザーのパルス幅とピーク出力パルスエネルギー面積に相当が同じ場合パルス幅が短いほどピーク出力は高い図 5 近赤外超短パルスレーザーを用いた雌性配偶体細胞の破壊実験シロイヌナズナの卵細胞特異的に Ca2+ センサー YC3.60 Nagai et al を発現させている青から赤にかけて赤いほど Ca2+ 濃度が高いことを示す左に胚珠と雌性配偶体を構成する細胞の模式図を示す A 助細胞の細胞膜付近にパルスレーザーを照射雷マークすることにより助細胞を破壊したレーザー照射後 30 秒で卵細胞内に Ca2+ 濃度の一過的な上昇が観察される B 中央細胞の細胞膜付近にパルスレーザーを照射雷マークすることにより中央細胞を破壊した照射後 5 分経過しても卵細胞内の Ca2+ 濃度変化は観察されないスケールバーは 10 μm Hamamura et al より改変けを破壊できることを示唆しているまたオオカナダモうに近赤外超短パルスレーザーを用いてレーザー照射部の生細胞において 800 nm のフェムト秒パルスレーザー位を変化させることで細胞全体を破壊するかあるいはを葉緑体の一部分に照射することにより原形質流動など細胞活性を損なうことなく特定のオルガネラのみを破壊すの細胞活性に影響を与えることなく葉緑体ひとつのみのるかなど精密な光顕微操作が可能となっている破壊にも成功している Tirlapur and König 2002a このよそれでは具体的な実施例を見ていこう Hamamura et al.

6 Vol. 49, No. 2, 図 6 CALI BY - 2 GFP - Fimbrin 50 μm 2014 Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ 5 UV 800 nm 5A Ca 2+ Ca 2+ 5B Ca 2+ Hamamura et al Ca 近赤外超短パルスレーザーによる CALI

7 102 Vol. 49, No. 2, 2014 Chromophore - assisted light inactivation ; CALI ROS Sano et al Jay 1988 ; Surrey et al CALI McLean et al CALI CALI Tanabe et al CALI Hasegawa et al. in press BY - 2 CALI BY - 2 Fimbrin GFP GFP - Fimbrin BY - 2 Sano et al nm 6 CALI おわりに 2 GaAsP CALI GFP CALI ROS KillerRed minisog SuperNova Bulina et al ; Qi et al ; Take moto et al Kurihara et al 謝辞 Rita Groß - Hardt JST ERATO 文石館文善 2011 pp 細川陽一郎水多陽子栗原大輔東山哲也 in vivo Plant Morph 26 : Bargmann, CI and Avery, L 1995 Laser killing of cells in Caenorhabditis elegans. Methods Cell Biol 48 : Benninger, RKP and Piston, DW 2013 Two - photon excitation microscopy for the study of living cells and tissues. Curr Protoc Cell Biol Chapter 4 : Unit Bulina, ME, Chudakov, DM, Britanova, OV, Yanushevich, YG, Staroverov, DB, Chepurnykh, TV, Merzlyak, EM, Shkrob, MA, Lukyanov, S and Lukyanov, KA 2006 A genetically encoded photosensitizer. Nat Biotechnol 24 : Cheung, AY, Boavida, LC, Aggarwal, M, Wu, HM and Feijó, JA 2010 The pollen tube journey in the pistil and imaging the in vivo process by two - photon microscopy. J Exp Bot 61 : Colaço, R, Moreno, N and Feijó, JA 2012 On the fast lane : mitochon dria structure, dynamics and function in growing pollen tubes. J Microsc 247 : Feijó, JA and Moreno, N 2004 Imaging plant cells by two - photon excitation. Protoplasma 223 : Hamamura, Y, Saito, C, Awai, C, Kurihara, D, Miyawaki, A, Nakagawa, T, Kanaoka, MM, Sasaki, N, Nakano, A, Berger, F and Higashiyama, T 2011 Live - cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana. Curr Biol 21 : Hamamura, Y, Nishimaki, M, Takeuchi, H, Geitmann, A, Kurihara, D and Higashiyama, T 2014 Live imaging of calcium spikes during double fertilization in Arabidopsis. Nat Commun 5 : Hasegawa, J, Higaki, T, Hamamura, Y, Kurihara, D, Kutsuna, N, Higashiyama, T, Hasezawa, S and Matsunaga, S in press Vacuole subdivision in plant cell growth by the genotoxic stress inducing DNA double strand breaks. Cytologia. 献

8 Vol. 49, No. 2, Hepler, PK and Gunning, BES 1998 Confocal fluorescence microscopy of plant cells. Protoplasma 201 : Helmchen, F and Denk, W 2005 Deep tissue two - photon microscopy. Nat Methods 2 : Higashiyama, T, Yabe, S, Sasaki, N, Nishimura, YSM, Kuroiwa, H and Kuroiwa, T 2001 Pollen tube attraction by the synergid cell. Science 293 : Jay, DG 1988 Selective destruction of protein function by chromophore - assisted laser inactivation. Proc Natl Acad Sci USA 85 : Kawakami, R, Sawada, K, Sato, A, Hibi, T, Kozawa, Y, Sato, S, Yokoyama, H and Nemoto, T 2013 Visualizing hippocampal neurons with in vivo two - photon microscopy using a 1030 nm picosecond pulse laser. Sci Rep 3 : König, K, Riemann, I and Fritzsche, W 2001 Nanodissection of human chromosomes with near - infrared femtosecond laser pulses. Opt Lett 26 : Kurihara, D, Hamamura, Y and Higashiyama, T 2013 Live - cell analysis of plant reproduction : live - cell imaging, optical manipulation, and advanced microscopy technologies. Dev Growth Differ 55 : McLean, MA, Rajfur, Z, Chen, Z, Humphrey, D, Yang, B, Sligar, SG and Jacobson, K 2009 Mechanism of chromophore assisted laser inactivation employing fluorescent proteins. Anal Chem 81 : Miller, MJ, Wei, SH, Parker, I and Cahalan, MD 2002 Two - photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296 : Müller, SM, Galliardt, H, Schneider, J, Barisas, BG and Seidel, T 2013 Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells. Front Plant Sci 4 : 413. Moreno, N, Bougourd, S, Haseloff, J and Feijó, JA 2006 Imaging plant cells. Handbook of Biological Confocal Microscopy, Ed. Pawley, JB, pp SpringerScience+Business Media, New York. Nagai, T, Yamada, S, Tominaga, T, Ichikawa, M and Miyawaki, A 2004 Expanded dynamic range of fluorescent indicators for Ca 2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci USA 101 : Okuda, S, Tsutsui, H, Shiina, K, Sprunck, S, Takeuchi, H, Yui, R, Kasahara, RD, Hamamura, Y, Mizukami, A, Susaki, D, Kawano, N, Sakakibara, T, Namiki, S, Itoh, K, Otsuka, K, Matsuzaki, M, Nozaki, H, Kuroiwa, T, Nakano, A, Kanaoka, MM, Dresselhaus, T, Sasaki, N and Higashiyama, T 2009 Defensin - like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458 : Qi, YB, Garren, EJ, Shu, X, Tsien, RY and Jin, Y 2012 Photo - inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein minisog. Proc Natl Acad Sci USA 109 : Sano, T, Higaki, T, Oda, Y, Hayashi, T and Hasezawa, S 2005 Appearance of actin microfilament twin peaks in mitosis and their function in cell plate formation, as visualized in tobacco BY - 2 cells expressing GFP - fimbrin. Plant J 44 : Sano, Y, Watanabe, W and Matsunaga, S 2014 Chromophore - assisted laser inactivation - towards a spatiotemporal - functional analysis of proteins, and the ablation of chromatin, organelle and cell function. J Cell Sci 127 : Stosiek, C, Garaschuk, O, Holthoff, K and Konnerth, A 2003 In vivo two - photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA 100 : Surrey, T, Elowitz, MB, Wolf, PE, Yang, F, Nédélec, F, Shokat, K and Leibler, S 1998 Chromophore - assisted light inactivation and self - organization of microtubules and motors. Proc Natl Acad Sci USA 95 : Takemoto, K, Matsuda, T, Sakai, N, Fu, D, Noda, M, Uchiyama, S, Kotera, I, Arai, Y, Horiuchi, M, Fukui, K, Ayabe, T, Inagaki, F, Suzuki, H and Nagai, T 2013 SuperNova, a monomeric photosensitizing fluorescent protein for chromophore - assisted light inactivation. Sci Rep 3 : Tanabe, T, Oyamada, M, Fujita, K, Dai, P, Tanaka, H and Takamatsu, T 2005 Multiphoton excitation - evoked chromophore - assisted laser inactivation using green fluorescent protein. Nat Methods 2 : Tirlapur, UK and König, K 2002a Femtosecond near - infrared laser pulses as a versatile non - invasive tool for intra - tissue nanoprocessing in plants without compromising viability. Plant J 31 : Tirlapur, UK and König, K 2002b Targeted transfection by femtosecond laser. Nature 418 : Truernit, E, Bauby, H, Dubreucq, B, Grandjean, O, Runions, J, Barthélémy, J and Palauqui, JC 2008 High - resolution whole - mount imaging of three - dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20 : van den Berg, C, Willemsen, V, Hendriks, G, Weisbeek, P and Scheres, B 1997 Short - range control of cell differentiation in the Arabidopsis root meristem. Nature 390 : Yoshida, S, Barbier de Reuille, P, Lane, B, Bassel, GW, Prusinkiewicz, P, Smith, RS and Weijers, D 2014 Genetic control of plant development by overriding a geometric division rule. Dev Cell 29 : : Tel : Fax : mizuta.yoko@f.mbox.nagoya - u.ac.jp Tel : Fax : kuri@bio.nagoya - u.ac.jp

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37-4.indd ISSN 0916-3328 東京大学アイソトープ総合センター VOL. 37 NO. 4 2007. 3. 26 アイソトープ総合センターの将来展望 40 18 18 20 3 800 1/3 1300 14 18 20 3 19 2 生きた細胞内の分子過程を時間空間計測する蛍光プローブの開発 1. はじめに Fura-2 Fura-2 198 1, 2 3, 4 2. 生体脂質分子の機能とその分析