Skip to main content
SpringerLink
Log in

Application of FLIM-FIDSAM for the in vivo analysis of hormone competence of different cell types

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Background fluorescence derived from subcellular compartments is a major drawback in high-resolution live imaging, especially of plant cells. A novel technique for contrast enhancement of fluorescence images of living cells expressing fluorescent fusion proteins termed fluorescence intensity decay shape analysis microscopy (FIDSAM) has been recently published and is applied here to plant cells expressing wild-type levels of a low-abundant membrane protein (BRI1-EGFP), demonstrating the applicability of FIDSAM to samples exhibiting about 80% autofluorescence. Furthermore, the combination of FIDSAM and fluorescence lifetime imaging microscopy enables the simultaneous determination and quantification of different ligand-specific responses in living cells with high spatial and temporal resolution even in samples with high autofluorescence background. Correlation of different responses can be used to determine the hormone ligand competence of different cell types as demonstrated here in BRI1-EGFP-expressing root and hypocotyl cells.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Westphal V, Rizzoli SO, Lauterbach MA, Kamin D, Jahn R, Hell SW (2008) Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320:246–249

    Article  CAS  Google Scholar 

  2. Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440:935–939

    Article  CAS  Google Scholar 

  3. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  CAS  Google Scholar 

  4. Schnell SA, Staines WA, Wessendor MW (1999) Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47:719–730

    CAS  Google Scholar 

  5. Elgass K, Caesar K, Schleifenbaum F, Stierhof Y-D, 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(Article No.):e5716

    Article  Google Scholar 

  6. Schleifenbaum F, Elgass K, Sackrow M, Caesar K, Berendzen K, Meixner AJ, Harter K (2009) 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 3:555–562. doi:10.1093/mp/ssp110

    Article  Google Scholar 

  7. 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. Proc SPIE 7568:756804

    Article  Google Scholar 

  8. Nakabayashi T, Wang HP, Kinjo M, Ohta N (2008) Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements. Photochem Photobiol Sci 7:668–670

    Article  CAS  Google Scholar 

  9. van Manen HJ, Verkuijlen P, Wittendorp P, Subramaniam V, van den Berg TK, Roos D, Otto C (2008) Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy. Biophys J 94:L67–L69

    Article  Google Scholar 

  10. Pepperkok R, Squire A, Geley S, Bastiaens PIH (1999) Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr Biol 9:269–272

    Article  CAS  Google Scholar 

  11. Billinton N, Knight AW (2001) Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal Biochem 291:175–197

    Article  CAS  Google Scholar 

  12. Suhling K, Davis DM, Phillips D (2002) The influence of solvent viscosity on the fluorescence decay and time-resolved anisotropy of green fluorescent protein. J Fluoresc 12:91–95

    Article  CAS  Google Scholar 

  13. Suhling K, Siegel J, Phillips D, French PMW, Leveque-Fort S, Webb SED, Davis DM (2002) Imaging the environment of green fluorescent protein. Biophys J 83:3589–3595

    Article  CAS  Google Scholar 

  14. Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, Berlin

    Book  Google Scholar 

  15. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    Article  CAS  Google Scholar 

  16. Wang ZY, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature 411:219–219

    Article  CAS  Google Scholar 

  17. Geldner N, Hyman DL, Wang XL, Schumacher K, Chory J (2007) Endosomal signaling of plant steroid receptor kinase BRI1. Genes Dev 21:1598–1602

    Article  CAS  Google Scholar 

  18. Clouse SD, Sasse JM (1998) Brassinosteroids: Essential regulators of plant growth and development. Annu Rev Plant Physiol Plant Mol Biol 49:427–451

    Article  CAS  Google Scholar 

  19. Sanchez-Rodriguez C, Rubio-Somoza I, Sibout R, Persson S (2010) Phytohormones and the cell wall in Arabidopsis during seedling growth. Trends Plant Sci 15:291–301

    Article  CAS  Google Scholar 

  20. Caesar K, Elgass K, Chen Z, Huppenberger P, Schleifenbaum F, Oecking C, Meixner AJ, Blatt MR, and Harter K (2010) A short brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana. Plant Journal (in press).

  21. Medina J, Ballesteros ML, Salinas J (2007) Phylogenetic and functional analysis of Arabidopsis RCI2 genes. J Exp Bot 58:4333–4346

    Article  CAS  Google Scholar 

  22. Medina J, Catala R, Salinas J (2001) Developmental and stress regulation of RCI2A and RCI2B, two cold-inducible genes of Arabidopsis encoding highly conserved hydrophobic proteins. Plant Physiol 125:1655–1666

    Article  CAS  Google Scholar 

  23. Mitsuya S, Taniguchi M, Miyake H, Takabe T (2005) Disruption of RCI2A leads to over-accumulation of Na+ and increased salt sensitivity in Arabidopsis thaliana plants. Planta 222:1001–1009

    Article  CAS  Google Scholar 

  24. Mitsuya S, Taniguchi M, Miyake H, Takabe T (2006) Overexpression of RC12A decreases Na+ uptake and mitigates salinity-induced damages in Arabidopsis thaliana plants. Physiol Plant 128:95–102

    Article  CAS  Google Scholar 

  25. Navarre C, Goffeau A (2000) Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane. EMBO J 19:2515–2524

    Article  CAS  Google Scholar 

  26. Nylander M, Heino P, Helenius E, Palva ET, Ronne H, Welin BV (2001) The low-temperature- and salt-induced RCI2A gene of Arabidopsis complements the sodium sensitivity caused by a deletion of the homologous yeast gene SNA1. Plant Mol Biol 45:341–352

    Article  CAS  Google Scholar 

  27. Elgass K, Caesar K, Harter K, Meixner AJ, and Schleifenbaum F (2010) Combining ocFLIM and FIDSAM reveals fast and dynamic physiological responses at subcellular resolution in living plant cells. Journal of Microscopy.

  28. Xu WH, Huang J, Li BH, Li JY, Wang YH (2008) Is kinase activity essential for biological functions of BRI1? Cell Res 18:472–478

    Article  CAS  Google Scholar 

  29. Wang XL, Chory J (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313:1118–1122

    Article  CAS  Google Scholar 

  30. Tang WQ, Kim TW, Oses-Prieto JA, Sun Y, Deng ZP, Zhu SW, Wang RJ, Burlingame AL, Wang ZY (2008) BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis. Science 321:557–560

    Article  CAS  Google Scholar 

  31. Otegui MS, Spitzer C (2008) Endosomal functions in plants. Traffic 9:1589–1598

    Article  CAS  Google Scholar 

  32. Teale WD, Ditengou FA, Dovzhenko AD, Li X, Molendijk AM, Ruperti B, Paponov I, Palme K (2008) Auxin as a model for the integration of hormonal signal processing and transduction. Mol Plant 1:229–237

    Article  CAS  Google Scholar 

  33. Blum C, Stracke F, Becker S, Mullen K, Meixner AJ (2001) Discrimination and interpretation of spectral phenomena by room-temperature single-molecule spectroscopy. J Phys Chem A 105:6983–6990

    Article  CAS  Google Scholar 

  34. Schleifenbaum F, Blum C, Elgass K, Subramaniam V, Meixner AJ (2008) New Insights into the Photophysics of DsRed by Multiparameter Spectroscopy on Single Proteins. J Phys Chem B 112:7669–7674

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Karin Schumacher for providing the BRI1-GFP line and to Christian Blum for the purified eGFP. This work was supported by a DFG grant to K.H. (HA 2146/10-1) and doctoral and junior group leader fellowships of the state Baden-Württemberg and the University of Tübingen to K.C., K.E., and F.S.

Author information

Authors and Affiliations

  1. Institute of Physical and Theoretical Chemistry, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany

    Kirstin Elgass & Alfred J. Meixner

  2. Center for Plant Molecular Biology, Department of Plant Physiology, University of Tübingen, Auf der Morgenstelle 1, 72076, Tübingen, Germany

    Katharina Caesar, Dierk Wanke, Klaus Harter & Frank Schleifenbaum

Authors
  1. Kirstin Elgass
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katharina Caesar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dierk Wanke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Klaus Harter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alfred J. Meixner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Frank Schleifenbaum
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frank Schleifenbaum.

Additional information

Published in the special issue Optical Biochemical and Chemical Sensors (Europtrode X) with guest editor Jiri Homola.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 1115 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Elgass, K., Caesar, K., Wanke, D. et al. Application of FLIM-FIDSAM for the in vivo analysis of hormone competence of different cell types. Anal Bioanal Chem 398, 1919–1925 (2010). https://doi.org/10.1007/s00216-010-4127-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-010-4127-4

Keywords

Search

Navigation