Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 May;80(5):2396–2408. doi: 10.1016/S0006-3495(01)76209-1

Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy.

G S Harms 1, L Cognet 1, P H Lommerse 1, G A Blab 1, T Schmidt 1
PMCID: PMC1301428  PMID: 11325739

Abstract

The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.

Full Text

The Full Text of this article is available as a PDF (401.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baird G. S., Zacharias D. A., Tsien R. Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11984–11989. doi: 10.1073/pnas.97.22.11984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Basché T, Moerner WE, Orrit M, Talon H. Photon antibunching in the fluorescence of a single dye molecule trapped in a solid. Phys Rev Lett. 1992 Sep 7;69(10):1516–1519. doi: 10.1103/PhysRevLett.69.1516. [DOI] [PubMed] [Google Scholar]
  3. Benson R. C., Meyer R. A., Zaruba M. E., McKhann G. M. Cellular autofluorescence--is it due to flavins? J Histochem Cytochem. 1979 Jan;27(1):44–48. doi: 10.1177/27.1.438504. [DOI] [PubMed] [Google Scholar]
  4. Bruchez M., Jr, Moronne M., Gin P., Weiss S., Alivisatos A. P. Semiconductor nanocrystals as fluorescent biological labels. Science. 1998 Sep 25;281(5385):2013–2016. doi: 10.1126/science.281.5385.2013. [DOI] [PubMed] [Google Scholar]
  5. Chan W. C., Nie S. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science. 1998 Sep 25;281(5385):2016–2018. doi: 10.1126/science.281.5385.2016. [DOI] [PubMed] [Google Scholar]
  6. Creemers T. M., Lock A. J., Subramaniam V., Jovin T. M., Völker S. Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning. Nat Struct Biol. 1999 Jun;6(6):557–560. doi: 10.1038/9335. [DOI] [PubMed] [Google Scholar]
  7. De Giorgi F., Ahmed Z., Bastianutto C., Brini M., Jouaville L. S., Marsault R., Murgia M., Pinton P., Pozzan T., Rizzuto R. Targeting GFP to organelles. Methods Cell Biol. 1999;58:75–85. doi: 10.1016/s0091-679x(08)61949-4. [DOI] [PubMed] [Google Scholar]
  8. Dickson R. M., Cubitt A. B., Tsien R. Y., Moerner W. E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature. 1997 Jul 24;388(6640):355–358. doi: 10.1038/41048. [DOI] [PubMed] [Google Scholar]
  9. Eigen M., Rigler R. Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5740–5747. doi: 10.1073/pnas.91.13.5740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Funatsu T., Harada Y., Tokunaga M., Saito K., Yanagida T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature. 1995 Apr 6;374(6522):555–559. doi: 10.1038/374555a0. [DOI] [PubMed] [Google Scholar]
  11. Garcia-Parajo M. F., Veerman J. A., Segers-Nolten G. M., de Grooth B. G., Greve J., van Hulst N. F. Visualising individual green fluorescent proteins with a near field optical microscope. Cytometry. 1999 Jul 1;36(3):239–246. doi: 10.1002/(sici)1097-0320(19990701)36:3<239::aid-cyto14>3.3.co;2-p. [DOI] [PubMed] [Google Scholar]
  12. Giebel K., Bechinger C., Herminghaus S., Riedel M., Leiderer P., Weiland U., Bastmeyer M. Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy. Biophys J. 1999 Jan;76(1 Pt 1):509–516. doi: 10.1016/s0006-3495(99)77219-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Griffin B. A., Adams S. R., Tsien R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science. 1998 Jul 10;281(5374):269–272. doi: 10.1126/science.281.5374.269. [DOI] [PubMed] [Google Scholar]
  14. Harms G. S., Sonnleitner M., Schütz G. J., Gruber H. J., Schmidt T. Single-molecule anisotropy imaging. Biophys J. 1999 Nov;77(5):2864–2870. doi: 10.1016/S0006-3495(99)77118-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heikal A. A., Hess S. T., Baird G. S., Tsien R. Y., Webb W. W. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11996–12001. doi: 10.1073/pnas.97.22.11996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Iwane A. H., Funatsu T., Harada Y., Tokunaga M., Ohara O., Morimoto S., Yanagida T. Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro. FEBS Lett. 1997 Apr 28;407(2):235–238. doi: 10.1016/s0014-5793(97)00359-1. [DOI] [PubMed] [Google Scholar]
  17. Kneen M., Farinas J., Li Y., Verkman A. S. Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J. 1998 Mar;74(3):1591–1599. doi: 10.1016/S0006-3495(98)77870-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kubitscheck U., Kückmann O., Kues T., Peters R. Imaging and tracking of single GFP molecules in solution. Biophys J. 2000 Apr;78(4):2170–2179. doi: 10.1016/S0006-3495(00)76764-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lacoste T. D., Michalet X., Pinaud F., Chemla D. S., Alivisatos A. P., Weiss S. Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9461–9466. doi: 10.1073/pnas.170286097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matz M. V., Fradkov A. F., Labas Y. A., Savitsky A. P., Zaraisky A. G., Markelov M. L., Lukyanov S. A. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol. 1999 Oct;17(10):969–973. doi: 10.1038/13657. [DOI] [PubMed] [Google Scholar]
  21. Miyawaki A., Llopis J., Heim R., McCaffery J. M., Adams J. A., Ikura M., Tsien R. Y. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997 Aug 28;388(6645):882–887. doi: 10.1038/42264. [DOI] [PubMed] [Google Scholar]
  22. Moriyoshi K., Richards L. J., Akazawa C., O'Leary D. D., Nakanishi S. Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP. Neuron. 1996 Feb;16(2):255–260. doi: 10.1016/s0896-6273(00)80044-6. [DOI] [PubMed] [Google Scholar]
  23. Ormö M., Cubitt A. B., Kallio K., Gross L. A., Tsien R. Y., Remington S. J. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 1996 Sep 6;273(5280):1392–1395. doi: 10.1126/science.273.5280.1392. [DOI] [PubMed] [Google Scholar]
  24. Piston D. W., Patterson G. H., Knobel S. M. Quantitative imaging of the green fluorescent protein (GFP). Methods Cell Biol. 1999;58:31–48. doi: 10.1016/s0091-679x(08)61947-0. [DOI] [PubMed] [Google Scholar]
  25. Rinia H. A., Kik R. A., Demel R. A., Snel M. M., Killian J. A., van Der Eerden J. P., de Kruijff B. Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers. Biochemistry. 2000 May 16;39(19):5852–5858. doi: 10.1021/bi000010c. [DOI] [PubMed] [Google Scholar]
  26. Sako Y., Minoghchi S., Yanagida T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol. 2000 Mar;2(3):168–172. doi: 10.1038/35004044. [DOI] [PubMed] [Google Scholar]
  27. Saxton M. J., Jacobson K. Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct. 1997;26:373–399. doi: 10.1146/annurev.biophys.26.1.373. [DOI] [PubMed] [Google Scholar]
  28. Schmidt T., Schütz G. J., Baumgartner W., Gruber H. J., Schindler H. Imaging of single molecule diffusion. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2926–2929. doi: 10.1073/pnas.93.7.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schwille P., Haupts U., Maiti S., Webb W. W. Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys J. 1999 Oct;77(4):2251–2265. doi: 10.1016/S0006-3495(99)77065-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schwille P., Kummer S., Heikal A. A., Moerner W. E., Webb W. W. Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):151–156. doi: 10.1073/pnas.97.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schütz G. J., Kada G., Pastushenko V. P., Schindler H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 2000 Mar 1;19(5):892–901. doi: 10.1093/emboj/19.5.892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Swaminathan R., Hoang C. P., Verkman A. S. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys J. 1997 Apr;72(4):1900–1907. doi: 10.1016/S0006-3495(97)78835-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tsien R. Y. Fluorescent probes of cell signaling. Annu Rev Neurosci. 1989;12:227–253. doi: 10.1146/annurev.ne.12.030189.001303. [DOI] [PubMed] [Google Scholar]
  34. Tsien R. Y. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–544. doi: 10.1146/annurev.biochem.67.1.509. [DOI] [PubMed] [Google Scholar]
  35. Zühlke R. D., Pitt G. S., Deisseroth K., Tsien R. W., Reuter H. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature. 1999 May 13;399(6732):159–162. doi: 10.1038/20200. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES