Skip to main content
SpringerLink
Log in

Global Analysis of FRET–FLIM Data in Live Plant Cells

  • Protocol
  • First Online:
Fluorescence Spectroscopy and Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1076))

Abstract

This chapter describes the procedure for globally analyzing fluorescence lifetime imaging (FLIM) data for the observation and quantification of Förster resonance energy transfer (FRET) in live plant cells. The procedure is illustrated by means of a case study, for which plant protoplasts were transfected with different visible fluorescent proteins and subsequently imaged using two-photon excitation FLIM. Spatially resolved fluorescence lifetime images were obtained by application of global analysis using the program Glotaran, which is open-source and freely available software. Using this procedure it is possible to extract the fraction and distance of interacting species between, or conformational changes within proteins, from complex experimental FRET–FLIM datasets, even at low signal-to-noise ratios. In addition, the software allows excluding inherently present autofluorescence from the plant cells, which improves the accuracy of the FRET analysis. The results from the case study are presented and interpreted in the context of the current scientific understanding of these biological systems.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Borst JW, Visser AJWG (2010) Fluorescence lifetime imaging microscopy in life sciences. Meas Sci Technol 21:102002

    Article  Google Scholar 

  2. Beechem JM (1992) Global analysis of biochemical and biophysical data. Methods Enzymol 210:37–54

    Article  PubMed  CAS  Google Scholar 

  3. Bednarkiewicz A, Whelan MP (2008) Global analysis of microscopic fluorescence lifetime images using spectral segmentation and a digital micromirror spatial illuminator. J Biomed Opt 13:041316

    Article  PubMed  Google Scholar 

  4. Barber P, Ameer-Beg S, Gilbey J et al (2009) Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis. J R Soc Interface 6:S93–S105

    Article  CAS  Google Scholar 

  5. Wlodarczyk J, Woehler A, Kobe F et al (2008) Analysis of FRET signals in the presence of free donors and acceptors. Biophys J 94:986–1000

    Article  PubMed  CAS  Google Scholar 

  6. Laptenok SP, van Stokkum IHM, Borst JW et al (2012) Disentangling picosecond events that complicate the quantitative use of the calcium sensor YC3.60. J Phys Chem B 116:3013–3020

    Article  PubMed  CAS  Google Scholar 

  7. Snellenburg JJ, Laptenok SP, Seger R et al (2012) Glotaran: a Java-based graphical user interface for the R-package TIMP. J Stat Softw 49(3):1–23

    Google Scholar 

  8. Bücherl C, Aker J, de Vries S et al (2010) Probing protein-protein Interactions with FRET-FLIM. Methods Mol Biol 655:389–399

    Article  PubMed  Google Scholar 

  9. Russinova E, Borst JW, Kwaaitaal M et al (2004) Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16:3216–3229

    Article  PubMed  CAS  Google Scholar 

  10. Verveer PJ, Squire A, Bastiaens PIH (2000) Global analysis of fluorescence lifetime imaging microscopy data. Biophys J 78:2127–2137

    Article  PubMed  CAS  Google Scholar 

  11. Laptenok SP, Borst JW, Mullen KM et al (2010) Global analysis of Förster 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 

  12. Valeur B (2002) Molecular fluorescence: principles and applications. Wiley-VCH, New York

    Google Scholar 

  13. Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York

    Book  Google Scholar 

  14. Förster T (1949) Experimentelle und theoretische Untersuchung des zwischenmolekularen Ubergangs von Elektronenanregungsenergie. Z Naturforsch 4a:321–327

    Google Scholar 

  15. Laptenok SP, Mullen KM, Borst JW et al (2007) Fluorescence lifetime imaging microscopy (FLIM) data analysis with TIMP. J Stat Softw 18(8):1–20

    Article  Google Scholar 

  16. Klarenbeek JB, Goedhart J, Hink MA et al (2011) A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range. PLoS One 6:e19170

    Article  PubMed  CAS  Google Scholar 

  17. Goedhart J, van Weeren L, Hink MA et al (2010) Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7:137–139

    Article  PubMed  CAS  Google Scholar 

  18. Borst JW, Laptenok SP, Westphal AH 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 

  19. Wu F-H, Shen S-C, Lee L-Y et al (2009) Tape-Arabidopsis sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16

    Article  PubMed  Google Scholar 

  20. Press WH, Teukolsky SA, Vetterling WT et al (2007) Numerical recipes: the art of scientific computing, 3rd edn. Cambridge University Press, Cambridge, MA

    Google Scholar 

  21. Golub GH, Van Loan CF (1996) Matrix computations, 3rd edn. The Johns Hopkins University Press, Baltimore, MD

    Google Scholar 

  22. Laptenok SP, Visser NV, Engel R et al (2011) A general approach for detecting folding intermediates from steady-state and time-resolved fluorescence of single-tryptophan-containing proteins. Biochemistry 50:3441–3450

    Article  PubMed  CAS  Google Scholar 

  23. Krumova SB, Laptenok SP, Borst JW et al (2010) Monitoring photosynthesis in individual cells of Synechocystis sp. PCC 6803 on a picosecond timescale. Biophys J 99:2006–2015

    Article  PubMed  CAS  Google Scholar 

  24. Grinvald A, Steinberg IZ (1974) Analysis of fluorescence decay kinetics by method of least-squares. Anal Biochem 59:583–598

    Article  PubMed  CAS  Google Scholar 

  25. Vos K, van Hoek A, Visser AJWG (1987) Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics. Eur J Biochem 165:55–63

    Article  PubMed  CAS  Google Scholar 

  26. Boens N, Ameloot M, Yamazaki I et al (1988) On the use and the performance of the delta-function convolution method for the estimation of fluorescence decay parameters. Chem Phys 121:73–86

    Article  CAS  Google Scholar 

  27. Mullen KM, van Stokkum IHM (2007) TIMP: an R package for modeling multi-way spectroscopic measurements. J Stat Softw 18(3):1–46

    Article  Google Scholar 

  28. Kremers G-J, Hazelwood KL, Murphy CS et al (2009) Photoconversion in orange and red fluorescent proteins. Nat Methods 6:355–358

    Article  PubMed  CAS  Google Scholar 

  29. McAnaney TB, Zeng W, Doe CFE et al (2005) Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism. Biochemistry 44:5510–5524

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire d’Optique et Biosciences, INSERM U696-CNRS UMR7645, Ecole Polytechnique, Palaiseau, France

    Sergey P. Laptenok

  2. Faculty of Sciences, Department of Physics and Astronomy, VU University Amsterdam, Amsterdam, The Netherlands

    Joris J. Snellenburg

  3. Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands

    Christoph A. Bücherl

  4. Molecular Plant Physiology and Biophysics, Julius-Maximilians-University Würzburg, Würzburg, Germany

    Kai R. Konrad

  5. Laboratory of Biochemistry and Microspectroscopy Centre, Wageningen University, Wageningen, The Netherlands

    Jan Willem Borst

  6. Center for BioSystems Genomics, Wageningen, The Netherlands

    Jan Willem Borst

Authors
  1. Sergey P. Laptenok
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joris J. Snellenburg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christoph A. Bücherl
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kai R. Konrad
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jan Willem Borst
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. KULeuven-Biomolecular Dynamics BioSCENTer, Leuven, Belgium

    Yves Engelborghs

  2. MicroSpectroscopy Centre, University of Wageningen Biochemistry, Wageningen, The Netherlands

    Antonie J.W.G. Visser

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Laptenok, S.P., Snellenburg, J.J., Bücherl, C.A., Konrad, K.R., Borst, J.W. (2014). Global Analysis of FRET–FLIM Data in Live Plant Cells. In: Engelborghs, Y., Visser, A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology, vol 1076. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-649-8_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-649-8_21

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-648-1

  • Online ISBN: 978-1-62703-649-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation