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Fluorescence Microscopy

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Arabidopsis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1062))

Abstract

Optical microscopy has developed as an indispensable tool for Arabidopsis cell biology. This is due to the high sensitivity, good spatial resolution, minimal invasiveness, and availability of autofluorescent proteins, which can be specifically fused to a distinct protein of interest. In this chapter, we introduce the theoretical concepts of fluorescence emission necessary to accomplish quantitative and functional cell biology using optical microscopy. The main focus lies on spectroscopic techniques, which, in addition to intensity-based studies, provide functional insight into cellular processes.

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References

  1. Stephens DJ, Allan VJ (2003) Light microscopy techniques for live cell imaging. Science 300:82–86

    Article  PubMed  CAS  Google Scholar 

  2. Lakowicz JR (2006) Principles of fluorescence spectroscopy. Kluwer, New York

    Book  Google Scholar 

  3. Schleifenbaum F, Blum C, Subramaniam V, Meixner AJ (2009) Single molecule spectral dynamics at room temperature. Mol Phys 107:1923–1942

    Article  CAS  Google Scholar 

  4. van Munster EB, Gadella TW (2005) Fluorescence lifetime imaging microscopy (FLIM). Adv Biochem Eng Biotechnol 95:143–175

    PubMed  Google Scholar 

  5. Ntziachristos V (2006) Fluorescence molecular imaging. Annu Rev Biomed Eng 8:1–33

    Article  PubMed  CAS  Google Scholar 

  6. Pepperkok R, Ellenberg J (2006) High-throughput fluorescence microscopy for systems biology. Nat Rev Mol Cell Biol 7:690–696

    Article  PubMed  CAS  Google Scholar 

  7. Suzuki T, Matsuzaki T, Hagiwara H, Aoki T, Takata K (2007) Recent advances in fluorescent labeling techniques for fluorescence microscopy. Acta Histochem Cytochem 40:131–137

    Article  PubMed  CAS  Google Scholar 

  8. Valeur B (2002) Molecular fluorescence: principles and applications. Wiley-WCH, Weinheim

    Google Scholar 

  9. Shotton DM (1989) Confocal scanning optical microscopy and its applications for biological specimens. J Cell Sci 97:175–206

    Google Scholar 

  10. Abbe E (1904) Abhandlungen über die Theorie des Mikroskops. Verlag G. Fischer, Jena

    Google Scholar 

  11. Axelrod D, Gerard M, Ian P (2003) Total internal reflection fluorescence microscopy in cell biology. In: Methods in enzymology. Academic Press 36:1–33. Biophotonics, Part B, Elsevier (Amsterdam). Editors: Gerard Marriot and Jan Parker

    Google Scholar 

  12. Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  PubMed  CAS  Google Scholar 

  13. Rust M, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–796

    Article  PubMed  CAS  Google Scholar 

  14. Heilemann M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47:6172–6176

    Article  CAS  Google Scholar 

  15. Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19:555–565

    Article  PubMed  CAS  Google Scholar 

  16. Hell S (2004) Strategy for far-field optical imaging and writing without diffraction limit. Phys Rev A 326:140–145

    CAS  Google Scholar 

  17. Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782

    Article  PubMed  CAS  Google Scholar 

  18. Dertinger T, Colyer R, Iyer G, Weiss S, Enderlein J (2009) Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc Natl Acad Sci 106:22287–22292

    Article  PubMed  CAS  Google Scholar 

  19. Esposito A, Wouters FS (2004) Fluorescence lifetime imaging microscopy. Curr Protoc Cell Biol Chapter 4:Unit 4.14

    Google Scholar 

  20. Elgass K, Caesar K, Schleifenbaum F, Stierhof Y-D, Meixner AJ, Harter K (2010) The fluorescence lifetime of BRI1-GFP as probe for the noninvasive determination of the membrane potential in living cells. SPIE Proc 7568:756838

    Google Scholar 

  21. Hille C et al (2008) Time-domain fluorescence lifetime imaging for intracellular pH sensing in living tissues. Anal Bioanal Chem 391:1871–1879

    Article  PubMed  CAS  Google Scholar 

  22. van Manen H-J et al (2008) Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy. Biophys J 94:L67–L69

    Article  PubMed  Google Scholar 

  23. Phillips D, Drake RC, O‘Connor DV, Christensen RL (1985) Time correlated single-photon counting (Tcspc) using laser excitation. Instrum Sci Technol 14:267–292

    Article  CAS  Google Scholar 

  24. Elgass K, Caesar K, Harter K, Meixner AJ, Schleifenbaum F (2011) Combining ocFLIM and FIDSAM reveals fast and dynamic physiological responses at subcellular resolution in living plant cells. J Microscopy 242(2):124–131

    Article  CAS  Google Scholar 

  25. Elgass K, Caesar K, Wanke D, Harter K, Meixner AJ, Schleifenbaum F (2010) Application of FLIM-FIDSAM for the in vivo analysis of hormone competence of different cell types. Anal Bioanal Chem 398:1919–1925

    Article  PubMed  CAS  Google Scholar 

  26. Schleifenbaum F et al (2010) Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging. Mol Plant 3:555–562

    Article  PubMed  CAS  Google Scholar 

  27. Schopfer P, Brennicke A (2010) Pflanzenphysiologie. Spektrum Akademischer Verlag, Heidleberg

    Google Scholar 

  28. Förster T (1948) Intermolecular energy migration and fluorescence. Ann Phys 2:55–75

    Article  Google Scholar 

  29. Grecco HE, Verveer PJ (2011) FRET in cell biology: still shining in the age of super-resolution? Chemphyschem 12:484–490

    Article  PubMed  CAS  Google Scholar 

  30. Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395

    Article  PubMed  CAS  Google Scholar 

  31. Jovin TM, Lidke DS, Jares-Erijman EA (2005) Fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). In: Evangelista V, Barsanti L, Passarelli V, Gualtieri P (eds) From cells to proteins: imaging nature across dimensions. Springer, Amsterdam, pp 209–216

    Chapter  Google Scholar 

  32. Schuler B, Eaton W (2008) Protein folding studied by single-molecule FRET. Curr Opin Struct Biol 18:16–26

    Article  PubMed  CAS  Google Scholar 

  33. Harter K, Meixner AJ, Schleifenbaum F (2011) Spectro-microscopy of living plant cells. Mol Plant. doi:10.1093/mp/ssr075

    Google Scholar 

  34. van Munster EB, Kremers GJ, Adjobo-Hermans MJ, Gadella TWJ (2005) Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching. J Microscopy 218:253–262

    Article  Google Scholar 

  35. Laptenok SP, Borst JW, Mullen KM, van Stokkum IHM, Visser AJWG, van Amerongen H (2010) Global analysis of Forster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence. Phys Chem Chem Phys 12:7593–7602

    Article  PubMed  CAS  Google Scholar 

  36. Borst JW et al (2008) Structural changes of yellow cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy. Biophys J 95:5399–5411

    Article  PubMed  CAS  Google Scholar 

  37. Visser A et al (2010) Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs. Eur Biophys J 39:241–253

    Article  PubMed  CAS  Google Scholar 

  38. Wanke D et al (2011) Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus. PLoS One 6:e16070

    Article  PubMed  CAS  Google Scholar 

  39. Schütze K, Harter K, Chaban C (2009) Bimolecular fluorescence complementation (BiFC): a novel tool to study protein-protein interactions in living plant cells. Methods Mol Biol 479:189–202

    Article  PubMed  Google Scholar 

  40. Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37:465–487

    Article  PubMed  CAS  Google Scholar 

  41. Hu C-D, Grinberg AV, Kerppola TK (2001). Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis. In: Current protocols in cell biology. John Wiley & Sons Inc, New York

    Google Scholar 

  42. Kapusta P, Wahl M, Benda A, Hof M, Enderlein J (2007) Fluorescence lifetime correlation spectroscopy. J Fluoresc 17:43–48

    Article  PubMed  CAS  Google Scholar 

  43. Reits EAJ, Neefjes JJ (2011) From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol 3:145–147

    Article  Google Scholar 

  44. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805

    Article  PubMed  CAS  Google Scholar 

  45. Chudakov DM, Lukyanov S, Lukyanov KA (2005) Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol 23:605–613

    Article  PubMed  CAS  Google Scholar 

  46. Cubitt A, Heim R, Adams S, Boyd A, Gross L, Tsien R (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455

    Article  PubMed  CAS  Google Scholar 

  47. Dixit R, Cyr R, Gilroy S (2006) Using intrinsically fluorescent proteins for plant cell imaging. Plant J 45:599–615

    Article  PubMed  CAS  Google Scholar 

  48. Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) Review—the fluorescent toolbox for assessing protein location and function. Science 312:217–224

    Article  PubMed  CAS  Google Scholar 

  49. Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572

    Article  PubMed  CAS  Google Scholar 

  50. Wachter R, Elsliger M, Kallio K, Hanson G, Remington J (1998) Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure 6:1267–1277

    Article  PubMed  CAS  Google Scholar 

  51. Lummer M et al (2011) Reversible photoswitchable DRONPA-s monitors nucleocytoplasmic transport of an RNA-binding protein in transgenic plants. Traffic 12:693–702

    Article  PubMed  CAS  Google Scholar 

  52. Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008) Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 56:505–516

    Article  PubMed  CAS  Google Scholar 

  53. Koushik SV, Chen H, Thaler C, Puhl HL III, Vogel SS (2006) Cerulean, Venus, and Venus Y67C FRET reference standards. Biophys J 91:L99–L101

    Article  PubMed  CAS  Google Scholar 

  54. Kneen M, Farinas J, Li Y, Verkman AS (1998) Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J 74:1591–1599

    Article  PubMed  CAS  Google Scholar 

  55. Elgass K, Caesar K, Schleifenbaum F, Stierhof YD, Meixner AJ, Harter K (2009) Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells. PLoS One 4:e5716

    Article  PubMed  Google Scholar 

  56. Hanson GT et al (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279:13044–13053

    Article  PubMed  CAS  Google Scholar 

  57. Belousov VV et al (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3:281–286

    Article  PubMed  CAS  Google Scholar 

  58. Miyawaki A, Griesbeck O, Heim R, Tsien R (1999) Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci U S A 96:2135–2140

    Article  PubMed  CAS  Google Scholar 

  59. Krebs M et al (2011) FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca2+ dynamics. Plant J. doi:10.1111/j.1365-313X.2011.04780.x

    PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Center for Plant Molecular Biology, University of Tuebingen, Tuebingen, Germany

    Sébastien Peter, Klaus Harter & Frank Schleifenbaum

  2. Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany

    Frank Schleifenbaum

Authors
  1. Sébastien Peter
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  2. Klaus Harter
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  3. Frank Schleifenbaum
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Editor information

Editors and Affiliations

  1. CSIC Centro Nacional de Biotecnología, Madrid, Spain

    Jose J. Sanchez-Serrano

  2. Depto. Biologia de Plantas, CSIC Madrid Centro de Investigaciones Biologicas, Madrid, Spain

    Julio Salinas

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Peter, S., Harter, K., Schleifenbaum, F. (2014). Fluorescence Microscopy. In: Sanchez-Serrano, J., Salinas, J. (eds) Arabidopsis Protocols. Methods in Molecular Biology, vol 1062. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-580-4_23

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  • DOI: https://doi.org/10.1007/978-1-62703-580-4_23

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-579-8

  • Online ISBN: 978-1-62703-580-4

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