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Protoplasma

, Volume 251, Issue 2, pp 383–394 | Cite as

FRET-FLIM applications in plant systems

  • Christoph A. Bücherl
  • Arjen Bader
  • Adrie H. Westphal
  • Sergey P. Laptenok
  • Jan Willem BorstEmail author
Special Issue: New/Emerging Techniques in Biological Microscopy

Abstract

A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Förster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.

Keywords

Fluorescence lifetime imaging microscopy (FLIM) Förster resonance energy transfer (FRET) Visible fluorescent protein (VFP) Global analysis Phasor plot analysis 

Abbreviations

ACR4

Arabidopsis crinkly 4

BiFC

Bimolecular fluorescence complementation

BRI1

Brassinosteroid insensitive 1

CLV1

Clavata1

CPK21

Calcium-dependent protein kinase 21

FCS

Fluorescence correlation spectroscopy

FCCS

Fluorescence cross-correlation spectroscopy

FLIM

Fluorescence lifetime imaging microscopy

FRET

Förster resonance energy transfer

FLS2

Flagellin sensing 2

GFP

Green fluorescent protein

LRR-RLK

Leucine-rich repeat receptor-like kinases

MFIS

Multiparameter fluorescence image spectroscopy

PM

Plasma membrane

SERK

Somatic embryogenesis receptor-like kinase

STED

Stimulated emission depletion

TCSPC

Time-correlated single photon counting

TIRF

Total internal reflection fluorescence

VFP

Visible fluorescent protein

Notes

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Bader AN, Hofman EG, Voortman J, Henegouwen PMPVE, Gerritsen HC (2009) Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution. Biophys J 97(9):2613–2622PubMedCentralPubMedCrossRefGoogle Scholar
  2. Barber PR, Ameer-Beg SM, Gilbey J, Edens RJ, Ezike I, Vojnovic B (2005) Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM. Multiphoton Microsc Biomed Sci 5700:171–181Google Scholar
  3. Bastiaens PI, Squire A (1999) Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol 9(2):48–52PubMedCrossRefGoogle Scholar
  4. Beck M, Zhou J, Faulkner C, MacLean D, Robatzek S (2012) Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting. Plant Cell 24(10):4205–4219PubMedCentralPubMedCrossRefGoogle Scholar
  5. Becker W, Bergmann A, Konig K, Tirlapur U (2001) Picosecond fluorescence lifetime microscopy by TCSPC imaging. Multiphoton Microsc Biomed Sci 2(19):414–419CrossRefGoogle Scholar
  6. Becker W, Bergmann A, Hink MA, Konig K, Benndorf K, Biskup C (2004) Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc Res Tech 63(1):58–66PubMedCrossRefGoogle Scholar
  7. Becker W, Su B, Holub O, Weisshart K (2011) FLIM and FCS detection in laser-scanning microscopes: increased efficiency by GaAsP hybrid detectors. Microsc Res Tech 74(9):804–811PubMedGoogle Scholar
  8. Borst JW, Hink MA, van Hoek A, Visser AJ (2005) Effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins. J Fluoresc 15(2):153–160PubMedCrossRefGoogle Scholar
  9. Breusegem SY, Levi M, Barry NP (2006) Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy. Nephron Exp Nephrol 103(2):e41–e49PubMedCrossRefGoogle Scholar
  10. Bücherl C, Aker J, de Vries S, Borst JW (2010) Probing protein-protein interactions with FRET-FLIM. Methods Mol Biol 655:389–399PubMedCrossRefGoogle Scholar
  11. Bücherl CA, van Esse GW, Kruis A, Luchtenberg J, Westphal AH, Aker J, van Hoek A, Albrecht C, Borst JW, de Vries SC (2013) Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling. Plant Physiol 162(4):1911–1925PubMedCentralPubMedCrossRefGoogle Scholar
  12. Buurman EP, Sanders R, Draaijer A, Gerritsen HC, Vanveen JJF, Houpt PM, Levine YK (1992) Fluorescence lifetime imaging using a confocal laser scanning microscope. Scanning 14(3):155–159CrossRefGoogle Scholar
  13. Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90(3):1103–1163PubMedCrossRefGoogle Scholar
  14. Clayton AHA, Hanley QS, Verveer PJ (2004) Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. J Microsc Oxf 213:1–5CrossRefGoogle Scholar
  15. Crivat G, Taraska JW (2012) Imaging proteins inside cells with fluorescent tags. Trends Biotechnol 30(1):8–16PubMedCentralPubMedCrossRefGoogle Scholar
  16. Day RN, Davidson MW (2012) Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells. Bioessays 34(5):341–350PubMedCentralPubMedCrossRefGoogle Scholar
  17. Day RN, Schaufele F (2005) Imaging molecular interactions in living cells. Mol Endocrinol 19(7):1675–1686PubMedCentralPubMedCrossRefGoogle Scholar
  18. De Rybel B, Moller B, Yoshida S, Grabowicz I, Barbier de Reuille P, Boeren S, Smith RS, Borst JW, Weijers D (2013) A bHLH complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis. Dev Cell 24(4):426–437PubMedCrossRefGoogle Scholar
  19. Dehmelt L, Bastiaens PI (2010) Spatial organization of intracellular communication: insights from imaging. Nat Rev Mol Cell Biol 11(6):440–452PubMedCrossRefGoogle Scholar
  20. Demir F, Horntrich C, Blachutzik JO, Scherzer S, Reinders Y, Kierszniowska S, Schulze WX, Harms GS, Hedrich R, Geiger D, Kreuzer I (2013) Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3. Proc Natl Acad Sci U S A 110(20):8296–8301PubMedCentralPubMedCrossRefGoogle Scholar
  21. Digman MA, Caiolfa VR, Zamai M, Gratton E (2008) The phasor approach to fluorescence lifetime imaging analysis. Biophys J 94(2):L14–L16PubMedCentralPubMedCrossRefGoogle Scholar
  22. Dowling K, Hyde SCW, Dainty JC, French PMW, Hares JD (1997) 2-D fluorescence lifetime imaging using a time-gated image intensifier. Opt Commun 135(1–3):27–31CrossRefGoogle Scholar
  23. Ehrhardt D (2003) GFP technology for live cell imaging. Curr Opin Plant Biol 6(6):622–628PubMedCrossRefGoogle Scholar
  24. Elgass K, Caesar K, Schleifenbaum F, Meixner AJ, Harter K (2010) The fluorescence lifetime of BRI1-GFP as probe for the noninvasive determination of the membrane potential in living cells. Imaging Manip Anal Biomol Cells Tissues Viii 7568Google Scholar
  25. 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 Microsc 242(2):124–131PubMedCrossRefGoogle Scholar
  26. Farinas J, Verkman AS (1999) Receptor-mediated targeting of fluorescent probes in living cells. J Biol Chem 274(12):7603–7606PubMedCrossRefGoogle Scholar
  27. Fereidouni F, Esposito A, Blab GA, Gerritsen HC (2011) A modified phasor approach for analyzing time-gated fluorescence lifetime images. J Microsc 244(3):248–258PubMedCrossRefGoogle Scholar
  28. Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 437(1–2):55–75CrossRefGoogle Scholar
  29. Gadella TWJ, Jovin TM, Clegg RM (1993) Fluorescence lifetime imaging microscopy (FLIM)—spatial-resolution of microstructures on the nanosecond time-scale. Biophys Chem 48(2):221–239CrossRefGoogle Scholar
  30. Gerdes HH, Kaether C (1996) Green fluorescent protein: applications in cell biology. FEBS Lett 389(1):44–47PubMedCrossRefGoogle Scholar
  31. Gerritsen HC, Asselbergs MAH, Agronskaia AV, Van Sark WGJHM (2002) Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution. J Microsc Oxf 206:218–224CrossRefGoogle Scholar
  32. Gordon GW, Berry G, Liang XH, Levine B, Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74(5):2702–2713PubMedCentralPubMedCrossRefGoogle Scholar
  33. Gratton E, Breusegem S, Sutin J, Ruan Q, Barry N (2003) Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J Biomed Opt 8(3):381–390PubMedCrossRefGoogle Scholar
  34. Grecco HE, Roda-Navarro P, Verveer PJ (2009) Global analysis of time correlated single photon counting FRET-FLIM data. Opt Express 17(8):6493–6508PubMedCrossRefGoogle Scholar
  35. Harter K, Meixner AJ, Schleifenbaum F (2012) Spectro-microscopy of living plant cells. Mol Plant 5(1):14–26PubMedCrossRefGoogle Scholar
  36. Hink MA, Shah K, Russinova E, de Vries SC, Visser AJ (2008) Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization. Biophys J 94(3):1052–1062PubMedCentralPubMedCrossRefGoogle Scholar
  37. Jares-Erijman EA, Jovin TM (2006) Imaging molecular interactions in living cells by FRET microscopy. Curr Opin Chem Biol 10(5):409–416PubMedCrossRefGoogle Scholar
  38. Kapusta P, Wahl M, Benda A, Hof M, Enderlein J (2007) Fluorescence lifetime correlation spectroscopy. J Fluoresc 17(1):43–48PubMedCrossRefGoogle Scholar
  39. Karpova TS, Baumann CT, He L, Wu X, Grammer A, Lipsky P, Hager GL, McNally JG (2003) Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J Micros Oxf 209:56–70CrossRefGoogle Scholar
  40. Kremers GJ, van Munster EB, Goedhart J, Gadella TWJ (2008) Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy. Biophys J 95(1):378–389PubMedCentralPubMedCrossRefGoogle Scholar
  41. Kwaaitaal M, Keinath NF, Pajonk S, Biskup C, Panstruga R (2010) Combined bimolecular fluorescence complementation and Forster resonance energy transfer reveals ternary SNARE complex formation in living plant cells. Plant Physiol 152(3):1135–1147PubMedCentralPubMedCrossRefGoogle Scholar
  42. Kwaaitaal M, Schor M, Hink MA, Visser AJ, de Vries SC (2011) Fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study receptor kinase mobility in planta. Methods Mol Biol 779:225–242PubMedCrossRefGoogle Scholar
  43. Lakowicz JR (1999) Principles of fluorescence spectroscopy (second edition). Springer, HeidelbergGoogle Scholar
  44. Laptenok SP, Borst JW, Mullen KM, van Stokkum IH, Visser AJ, 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(27):7593–7602PubMedCrossRefGoogle Scholar
  45. Laptenok SP, Snellenburg JJ, Bucherl CA, Konrad KR, Borst JW (2014) Global analysis of FRET-FLIM data in live plant cells. Methods Mol Biol 1076:481–502PubMedCrossRefGoogle Scholar
  46. Leung BO, Chou KC (2011) Review of super-resolution fluorescence microscopy for biology. Appl Spectrosc 65(9):967–980PubMedCrossRefGoogle Scholar
  47. Millis BA (2012) Evanescent-wave field imaging: an introduction to total internal reflection fluorescence microscopy. Methods Mol Biol 823:295–309PubMedCrossRefGoogle Scholar
  48. Morton PE, Parsons M (2011) Measuring FRET using time-resolved FLIM. Methods Mol Biol 769:403–413PubMedCrossRefGoogle Scholar
  49. Nair DK, Jose M, Kuner T, Zuschratter W, Hartig R (2006) FRET-FLIM at nanometer spectral resolution from living cells. Opt Express 14(25):12217–12229PubMedCrossRefGoogle Scholar
  50. Nakabayashi T, Oshita S, Sumikawa R, Sun F, Kinjo M, Ohta N (2012) pH dependence of the fluorescence lifetime of enhanced yellow fluorescent protein in solution and cells. J Photochem Photobiol Chem 235:65–71CrossRefGoogle Scholar
  51. Ntoukakis V, Schwessinger B, Segonzac C, Zipfel C (2011) Cautionary notes on the use of C-terminal BAK1 fusion proteins for functional studies. Plant Cell 23(11):3871–3878PubMedCentralPubMedCrossRefGoogle Scholar
  52. Peter M, Ameer-Beg SM, Hughes MK, Keppler MD, Prag S, Marsh M, Vojnovic B, Ng T (2005) Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophys J 88(2):1224–1237PubMedCentralPubMedCrossRefGoogle Scholar
  53. Redford GI, Clegg RM (2005) Polar plot representation for frequency-domain analysis of fluorescence lifetimes. J Fluoresc 15(5):805–815PubMedCrossRefGoogle Scholar
  54. Ries J, Kaplan C, Platonova E, Eghlidi H, Ewers H (2012) A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9(6):582–584PubMedCrossRefGoogle Scholar
  55. Russinova E, Borst JW, Kwaaitaal M, Cano-Delgado A, Yin Y, Chory J, de Vries SC (2004) Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16(12):3216–3229PubMedCentralPubMedCrossRefGoogle Scholar
  56. Schleifenbaum F, Elgass K, Sackrow M, Caesar K, Berendzen K, Meixner AJ, Harter K (2010) Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging: a novel technique for fluorescence microscopy of endogenously expressed fusion-proteins. Mol Plant 3(3):555–562PubMedCrossRefGoogle Scholar
  57. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909PubMedCrossRefGoogle Scholar
  58. Siegel J, Elson DS, Webb SE, Lee KC, Vlandas A, Gambaruto GL, Leveque-Fort S, Lever MJ, Tadrous PJ, Stamp GW, Wallace AL, Sandison A, Watson TF, Alvarez F, French PM (2003) Studying biological tissue with fluorescence lifetime imaging: microscopy, endoscopy, and complex decay profiles. Appl Opt 42(16):2995–3004PubMedCrossRefGoogle Scholar
  59. Stahl Y, Grabowski S, Bleckmann A, Kuhnemuth R, Weidtkamp-Peters S, Pinto KG, Kirschner GK, Schmid JB, Wink RH, Hulsewede A, Felekyan S, Seidel CA, Simon R (2013) Moderation of Arabidopsis root stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr Biol 23(5):362–371PubMedCrossRefGoogle Scholar
  60. Sun Y, Day RN, Periasamy A (2011) Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Nat Protoc 6(9):1324–1340PubMedCentralPubMedCrossRefGoogle Scholar
  61. Thaler C, Koushik SV, Blank PS, Vogel SS (2005) Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophys J 89(4):2736–2749PubMedCentralPubMedCrossRefGoogle Scholar
  62. van Munster EB, Gadella TW (2005) Fluorescence lifetime imaging microscopy (FLIM). Adv Biochem Eng Biotechnol 95:143–175PubMedGoogle Scholar
  63. Visser AJ, Laptenok SP, Visser NV, van Hoek A, Birch DJ, Brochon JC, Borst JW (2010) Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs. Eur Biophys J 39(2):241–253PubMedCrossRefGoogle Scholar
  64. Wombacher R, Cornish VW (2011) Chemical tags: applications in live cell fluorescence imaging. J Biophotonics 4(6):391–402PubMedCrossRefGoogle Scholar
  65. Wouters FS, Bastiaens PI (2001) Imaging protein-protein interactions by fluorescence resonance energy transfer (FRET) microscopy. Curr Protoc Cell Biol Chapter 17:Unit 17.1Google Scholar
  66. Xia ZP, Liu YH (2001) Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J 81(4):2395–2402PubMedCentralPubMedCrossRefGoogle Scholar
  67. Yasuda R (2006) Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr Opin Neurobiol 16(5):551–561PubMedCrossRefGoogle Scholar
  68. Zelazny E, Borst JW, Muylaert M, Batoko H, Hemminga MA, Chaumont F (2007) FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization. Proc Natl Acad Sci U S A 104(30):12359–12364PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Christoph A. Bücherl
    • 1
  • Arjen Bader
    • 2
  • Adrie H. Westphal
    • 4
  • Sergey P. Laptenok
    • 3
  • Jan Willem Borst
    • 4
    Email author
  1. 1.The Sainsbury LaboratoryNorwich Research ParkNorwichUK
  2. 2.Laboratory of Biophysics and Microspectroscopy CentreWageningen UniversityWageningenThe Netherlands
  3. 3.School of ChemistryUniversity of East AngliaNorwichUK
  4. 4.Laboratory of Biochemistry and Microspectroscopy CentreWageningen UniversityWageningenThe Netherlands

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