Skip to main content
SpringerLink
Log in

Monitoring dynamic systems with multiparameter fluorescence imaging

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

Abstract

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) Science 312:217–224

    Article  CAS  Google Scholar 

  2. Xie XS, Yu J, Yang WY (2006) Science 312:228–230

    Article  CAS  Google Scholar 

  3. Wouters FS, Verveer PJ, Bastiaens PIH (2001) Trends Cell Biol 11:203–211

    Article  CAS  Google Scholar 

  4. Truong K, Ikura M (2001) Curr Opin Struct Biol 11:573–578

    Article  CAS  Google Scholar 

  5. Sekar RB, Periasamy A (2003) J Cell Biol 160:629–633

    Article  CAS  Google Scholar 

  6. Meyer BH, Segura JM, Martinez KL, Hovius R, George N, Johnsson K, Vogel H (2006) Proc Natl Acad Sci USA 103:2138–2143

    Article  CAS  Google Scholar 

  7. Hoppe A, Christensen K, Swanson JA (2002) Biophys J 83:3652–3664

    CAS  Google Scholar 

  8. Bastiaens PIH, Squire A (1999) Trends Cell Biol 9:48–52

    Article  CAS  Google Scholar 

  9. Jares-Erijman EA, Jovin TM (2003) Nature Biotechnol 21:1387–1395

    Article  CAS  Google Scholar 

  10. Clayton AH, Hanley QS, Arndt-Jovin DJ, Subramaniam V, Jovin TM (2002) Biophys J 83:1631–1649

    CAS  Google Scholar 

  11. Levi V, Ruan Q, Kis-Petikova K, Gratton E (2003) Biochem Soc Trans 31:997–1000

    CAS  Google Scholar 

  12. Seisenberger G, Ried MU, Endreß T, Büning H, Hallek M, Bräuchle C (2001) Science 294:1929–1932

    Article  CAS  Google Scholar 

  13. Schütz GJ, Axmann M, Freudenthaler S, Schindler H, Kandror K, Roder JC, Jeromin A (2004) Microsc Res Tech 63:159–167

    Article  Google Scholar 

  14. Kues T, Dickmanns A, Lührmann R, Peters R, Kubitscheck U (2001) Proc Natl Acad Sci USA 98:12021–12026

    Article  CAS  Google Scholar 

  15. Thompson NL, Lieto AM, Allen NW (2002) Curr Opin Struct Biol 12:634–641

    Article  Google Scholar 

  16. Bacia K, Schwille P (2003) Methods 29:74–85

    Article  CAS  Google Scholar 

  17. Saffarian S, Elson EL (2003) Biophys J 84:2030–2042

    CAS  Google Scholar 

  18. Kohl T, Schwille P (2005) Adv Biochem Eng Biotechnol 95:107–142

    CAS  Google Scholar 

  19. Brock R, Hink MA, Jovin TM (1998) Biophys J 75:2547–2557

    CAS  Google Scholar 

  20. Schwille P, Haupts U, Maiti S, Webb WW (1999) Biophys J 77:2251–2265

    CAS  Google Scholar 

  21. Gennerich A, Schild D (2000) Biophys J 79:3294–3306

    CAS  Google Scholar 

  22. Skinner JP, Chen Y, Müller JD (2005) Biophys J 89:1288–1301

    Article  CAS  Google Scholar 

  23. Xiao Y, Buschmann V, Weston KD (2005) Anal Chem 77:36–46

    Article  CAS  Google Scholar 

  24. Ruan Q, Cheng MA, Levi M, Gratton E, Mantulin WW (2004) Biophys J 87:1260–1267

    Article  CAS  Google Scholar 

  25. Balaji J, Maiti S (2005) Microsc Res Tech 66:198–202

    Article  CAS  Google Scholar 

  26. Rocheleau JV, Wiseman PW, Petersen NO (2003) Biophys J 84:4011–4022

    CAS  Google Scholar 

  27. Digman MA, Sengupta P, Wiseman PW, Brown CM, Horwitz AR, Gratton E (2005) Biophys J 88:L33–L36

    Article  CAS  Google Scholar 

  28. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E (2005) Biophys J 89:1317–1327

    Article  CAS  Google Scholar 

  29. Hebert B, Costantino S, Wiseman PW (2005) Biophys J 88:3601–3614

    Article  CAS  Google Scholar 

  30. Kolin DL, Costantino S, Wiseman PW (2006) Biophys J 90:628–639

    Article  CAS  Google Scholar 

  31. Palo K, Brand L, Eggeling C, Jäger S, Kask P, Gall K (2002) Biophys J 83:605–618

    CAS  Google Scholar 

  32. Kask P, Palo K, Fay N, Brand L, Mets Ü, Ullmann D, Jungmann J, Pschorr J, Gall K (2000) Biophys J 78:1703–1713

    Article  CAS  Google Scholar 

  33. Kask P, Palo K, Ullmann D, Gall K (1999) Proc Natl Acad Sci USA 96:13756–13761

    Google Scholar 

  34. Palo K, Mets Ü, Jäger S, Kask P, Gall K (2000) Biophys J 79:2858–2866

    CAS  Google Scholar 

  35. Fries JR, Brand L, Eggeling C, Köllner M, Seidel CAM (1998) J Phys Chem A 102:6601–6613

    Article  CAS  Google Scholar 

  36. Becker W, Bergmann A, Haustein E, Petrasek Z, Schwille P, Biskup C, Kelbauskas L, Benndorf K, Klöcker N, Anhut T, Riemann I, König K (2006) Microsc Res Tech 69:186–195

    Article  CAS  Google Scholar 

  37. Widengren J, Kudryavtsev V, Antonik M, Berger S, Gerken M, Seidel CAM (2006) Anal Chem 78:2039–2050

    Article  CAS  Google Scholar 

  38. Edman L, Mets Ü, Rigler R (1996) Proc Natl Acad Sci USA 93:6710–6715

    Article  CAS  Google Scholar 

  39. Maus M, Cotlet M, Hofkens J, Gensch T, De Schryver FC, Schaffer J, Seidel CAM (2001) Anal Chem 73:2078–2086

    Article  CAS  Google Scholar 

  40. Bowen BP, Scruggs A, Enderlein J, Sauer M, Woodbury N (2004) J Phys Chem A 108:4799–4804

    Article  CAS  Google Scholar 

  41. Enderlein J, Sauer M (2001) J Phys Chem A 105:48–53

    Article  CAS  Google Scholar 

  42. Tellinghuisen J, Goodwin PM, Ambrose WP, Martin JC, Keller RA (1994) Anal Chem 66:64–72

    Article  CAS  Google Scholar 

  43. Enderlein J, Goodwin PM, van Orden A, Ambrose WP, Erdmann R, Keller RA (1997) Chem Phys Lett 270:464–470

    Article  CAS  Google Scholar 

  44. Schaffer J, Volkmer A, Eggeling C, Subramaniam V, Striker G, Seidel CAM (1999) J Phys Chem A 103:331–336

    Article  CAS  Google Scholar 

  45. Antonik M, Felekyan S, Gaiduk A, Seidel CAM (2006) J Phys Chem B 110:6970–6978

    Article  CAS  Google Scholar 

  46. Eggeling C, Berger S, Brand L, Fries JR, Schaffer J, Volkmer A, Seidel CAM (2001) J Biotechnol 86:163–180

    Article  CAS  Google Scholar 

  47. Rothwell PJ, Berger S, Kensch O, Felekyan S, Antonik M, Wöhrl BM, Restle T, Goody RS, Seidel CAM (2003) Proc Natl Acad Sci USA 100:1655–1660

    Article  CAS  Google Scholar 

  48. Gaiduk A, Kühnemuth R, Antonik M, Seidel CAM (2005) ChemPhysChem 6:976–983

    Article  CAS  Google Scholar 

  49. Zander C, Sauer M, Drexhage KH, Ko DS, Schulz A, Wolfrum J, Brand L, Eggeling C, Seidel CAM (1996) Appl Phys B 63:517–523

    CAS  Google Scholar 

  50. Eggeling C, Schaffer J, Seidel CAM, Korte J, Brehm G, Schneider S, Schrof W (2001) J Phys Chem A 105:3673–3679

    Article  CAS  Google Scholar 

  51. Kühnemuth R, Seidel CAM (2001) Single Mol 2:251–254

    Article  Google Scholar 

  52. Browning H, Hackney DD, Nurse P (2003) Nat Cell Biol 5:812–818

    Article  CAS  Google Scholar 

  53. Koshioka M, Saski K, Masuhara H (1995) Appl Spectrosc 49:224–228

    Article  CAS  Google Scholar 

  54. Felekyan S, Kühnemuth R, Kudryavtsev V, Sandhagen C, Becker W, Seidel CAM (2005) Rev Sci Instrum 76:083104

    Google Scholar 

  55. Busch KE, Brunner D (2004) Curr Biol 14:548–559

    Article  CAS  Google Scholar 

  56. Kerres A, Vietmeier-Decker C, Ortiz J, Karig I, Beuter C, Hegemann J, Lechner J, Fleig U (2004) Mol Biol Cell 15:5255–5267

    Article  CAS  Google Scholar 

  57. Striker G, Subramaniam V, Seidel CAM, Volkmer A (1999) J Phys Chem B 103:8612–8617

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Ursula Fleig and Christoph Beuter for their generous support. They provided the cells and prepared these cell samples for imaging. We thank Stanislav Kalinin for helping us to find appropriate and statistically stable parameters to describe noisy FCS curves. We thank Stefan Marawske for helping us to perform live cell measurements. We thank Alexander Gaiduk for helping us to establish MFD imaging. CS gratefully acknowledges financial support from the BMBF Biofuture grant 0311865 and the SFB 590, Heinrich-Heine-University Düsseldorf.

Author information

Authors and Affiliations

  1. Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany

    Volodymyr Kudryavtsev, Suren Felekyan, Anna K. Woźniak, Marcelle König, Carl Sandhagen, Ralf Kühnemuth, Claus A. M. Seidel & Filipp Oesterhelt

Authors
  1. Volodymyr Kudryavtsev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suren Felekyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anna K. Woźniak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcelle König
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carl Sandhagen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ralf Kühnemuth
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Claus A. M. Seidel
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Filipp Oesterhelt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Claus A. M. Seidel or Filipp Oesterhelt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kudryavtsev, V., Felekyan, S., Woźniak, A.K. et al. Monitoring dynamic systems with multiparameter fluorescence imaging. Anal Bioanal Chem 387, 71–82 (2007). https://doi.org/10.1007/s00216-006-0917-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-006-0917-0

Keywords

Search

Navigation