Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

Plant Methods

Jinxing Lin¹  Myung K Kim²  Huaiqin Hao¹  Lusheng Fan³  William M Ash²  Yinglang Wan¹ 

[1] Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China;Digital Holography and Microscopy Laboratory, Department of Physics, University of South Florida, Tampa, Florida 33620, USA;Gradual School of Chinese Academy of Sciences, Beijing, 100049, China

关键词: cell wall;    intact cell;    optical analysis;    VA-TIRFM;    Quantitative;   

Others  :  822977
DOI  :  10.1186/1746-4811-7-27

 received in 2011-07-24, accepted in 2011-09-24,  发布年份 2011

【 摘 要 】

Background

Total internal reflection fluorescence microscopy (TIRFM) is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM) was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology.

Results

Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein) and clathrin light chain (a vesicle coat protein) support our theoretical analysis.

Conclusions

These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.

【 授权许可】

   
2011 Wan et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Fish K: Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Cytom 2009, 12:Unit 12.18.
• [2]Groves JT, Parthasarathy R, Forstner MB: Fluorescence imaging of membrane dynamics. Annu Rev Biomed Eng 2008, 10:311-338.
• [3]Mashanov GI, Tacon D, Knight AE, Peckham M, Molloyc JE: Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. Methods 2003, 29:142-152.
• [4]Sako Y, Minoguchi S, Yanagida T: Single-molecule imaging of EGFR signaling on the surface of living cells. Nat Cell Biol 2000, 2:168-172.
• [5]Gutierrez R, Grossmann G, Frommer WB, Ehrhardt DW: Opportunities to explore plant membrane organization with super-resolution microscopy. Plant Physiol 2010, 154:463-466.
• [6]Tanner W, Malinsky J, Opekarová M: In plant and animal cells, detergent-resistant membranes do not define functional membrane rafts. Plant Cell 2011, 23:1191-1193.
• [7]Konopka CA, Bednarek SY: Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex. Plant J 2008, 53:186-196.
• [8]Konopka CA, Backues SK, Bednarek SY: Dynamics of Arabidopsis dynamin-related protein 1C and a clathrin light chain at the plasma membrane. Plant Cell 2008, 20:1363-1380.
• [9]Konopka CA, Bednarek SY: Comparison of the dynamics and functional redundancy of the arabidopsis dynamin-related isoforms DRP1A and DRP1C during plant development. Plant Physiol 2008, 147:1590-1602.
• [10]Woolley JT: Refractive index of soybean leaf cell walls. Plant Physiol 1975, 55:172-174.
• [11]Derbyshire P, Findlay K, McCann MC, Robert K: Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness. J Exp Bot 2007, 58:2079-2089.
• [12]Loerke D, Mettlen M, Yarar D, Jaqaman K, Jaqaman H, Danuser G, Schmid SL: Cargo and dynamin regulate clathrin-coated pit maturation. PLoS Biol 2009, 7:e1000057.
• [13]Zhang W, Jiang Y, Wang Q, Ma X, Xiao Z, Zuo W, Fang X, Chen Y: Single-molecule imaging reveals transforming growth factor-b-induced type II receptor dimerization. Proc Natl Acad Sci USA 2009, 106:15679-15683.
• [14]Rivière D: An optical study of thin interfacial liquid films using total reflection of light. Exp Fluids 1987, 5:349-354.
• [15]Ash WM III, Krzewina L, Kim MK: Quantitative imaging of cellular adhesion by total internal reflection holographic microscopy. Appl Opt 2009, 48:144-152.
• [16]Cleary AL: Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis of Tradescantia virginiana leaf epidermal cells. Protoplasma 2001, 215:21-34.
• [17]Wojtaszek P, Baluska F, Kasprowicz A, Luczak M, Volkmann D: Domain-specific mechanosensory transmission of osmotic and enzymatic cell wall disturbances to the actin cytoskeleton. Protoplasma 2007, 230:217-230.
• [18]Wan Y, Eisinger W, Ehrhardt D, Kubitscheck U, Baluska F, Briggs W: The subcellular localization and blue-light-induced movement of phototropin 1-GFP in etiolated seedlings of Arabidopsis thaliana. Mol Plant 2008, 1:103-117.
• [19]Kaiserli E, Sullivan S, Jones MA, Feeney KA, Christie JM: Domain swapping to assess the mechanistic basis of Arabidopsis phototropin 1 receptor kinase activation and endocytosis by blue light. Plant Cell 2009, 21:3226-3244.
• [20]Kirchhausen T: Imaging endocytic clathrin structures in living cells. Trends Cell Biol 2009, 19:596-605.
• [21]Ryan TA: Kiss-and-run, fuse-pinch-and-linger, fuse-and-collapse: The life and times of a neurosecretory granule. Proc Natl Acad Sci USA 2003, 100:2171-2173.
• [22]Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, Danuser G: Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 2008, 5:695-702.
• [23]Wang X, Teng Y, Wang Q, Li X, Sheng X, Zheng M, Šamaj J, Baluška F, Lin J: Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy. Plant Physiol 2006, 141:1591-1603.
• [24]Zheng M, Beck M, Müller J, Chen T, Wang X, Wang F, Wang Q, Wang Y, BalušKa F, Logan DC, Šamaj J, Lin J: Actin turnover is required for myosin-dependent mitochondrial movements in Arabidopsis root hairs. PLoS ONE 2009, 4:e5961.
• [25]Veliz LA, Toro CA, Vivar JP, Arias LA, Villegas J, Castro MA, Brauchi S: Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains. PLoS ONE 2010, 5:e13290.
• [26]Hern JA, Baig AH, Mashanov GI, Birdsall B, Corrie JE, Lazareno S, Molloy JE, Birdsall NJ: Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proc Natl Acad Sci USA 2010, 107:2693-2698.
• [27]Sakamoto K, Briggs WR: Cellular and subcellular localization of phototropin 1. Plant Cell 2002, 14:1723-1735.