Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Aug;79(2):1129–1138. doi: 10.1016/S0006-3495(00)76366-1

Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection.

D C Lamb 1, A Schenk 1, C Röcker 1, C Scalfi-Happ 1, G U Nienhaus 1
PMCID: PMC1301008  PMID: 10920042

Abstract

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure chemical reaction rates and diffusion coefficients of molecules in thermal equilibrium. The capabilities of FCS can be enhanced by measuring the energy, polarization, or delay time between absorption and emission of the collected fluorescence photons in addition to their arrival times. This information can be used to change the relative intensities of multiple fluorescent species in FCS measurements and, thus, the amplitude of the intensity autocorrelation function. Here we demonstrate this strategy using lifetime gating in FCS experiments. Using pulsed laser excitation and laser-synchronized gating in the detection channel, we suppress photons emitted within a certain time interval after excitation. Three applications of the gating technique are presented: suppression of background fluorescence, simplification of FCS reaction studies, and investigation of lifetime heterogeneity of fluorescently labeled biomolecules. The usefulness of this technique for measuring forward and backward rates of protein fluctuations in equilibrium and for distinguishing between static and dynamic heterogeneity makes it a promising tool in the investigation of chemical reactions and conformational fluctuations in biomolecules.

Full Text

The Full Text of this article is available as a PDF (116.8 KB).

Selected References

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

  1. Bismuto E., Gratton E., Sirangelo I., Irace G. Structure and dynamics of the acidic compact state of apomyoglobin by frequency-domain fluorometry. Eur J Biochem. 1993 Nov 15;218(1):213–219. doi: 10.1111/j.1432-1033.1993.tb18367.x. [DOI] [PubMed] [Google Scholar]
  2. Bismuto E., Irace G., Sirangelo I., Gratton E. Pressure-induced perturbation of ANS-apomyoglobin complex: frequency domain fluorescence studies on native and acidic compact states. Protein Sci. 1996 Jan;5(1):121–126. doi: 10.1002/pro.5560050115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonnet G., Krichevsky O., Libchaber A. Kinetics of conformational fluctuations in DNA hairpin-loops. Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8602–8606. doi: 10.1073/pnas.95.15.8602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen Y., Müller J. D., So P. T., Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J. 1999 Jul;77(1):553–567. doi: 10.1016/S0006-3495(99)76912-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eggeling C., Fries J. R., Brand L., Günther R., Seidel C. A. Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1556–1561. doi: 10.1073/pnas.95.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Haupts U., Maiti S., Schwille P., Webb W. W. Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13573–13578. doi: 10.1073/pnas.95.23.13573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kask P., Palo K., Ullmann D., Gall K. Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13756–13761. doi: 10.1073/pnas.96.24.13756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kask P., Piksarv P., Pooga M., Mets U., Lippmaa E. Separation of the rotational contribution in fluorescence correlation experiments. Biophys J. 1989 Feb;55(2):213–220. doi: 10.1016/S0006-3495(89)82796-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kettling U., Koltermann A., Schwille P., Eigen M. Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1416–1420. doi: 10.1073/pnas.95.4.1416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Magde D., Elson E. L., Webb W. W. Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers. 1974 Jan;13(1):29–61. doi: 10.1002/bip.1974.360130103. [DOI] [PubMed] [Google Scholar]
  12. Maiti S., Haupts U., Webb W. W. Fluorescence correlation spectroscopy: diagnostics for sparse molecules. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11753–11757. doi: 10.1073/pnas.94.22.11753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rauer B., Neumann E., Widengren J., Rigler R. Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor. Biophys Chem. 1996 Jan 16;58(1-2):3–12. doi: 10.1016/0301-4622(95)00080-1. [DOI] [PubMed] [Google Scholar]
  14. Schwille P., Bieschke J., Oehlenschläger F. Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies. Biophys Chem. 1997 Jun 30;66(2-3):211–228. doi: 10.1016/s0301-4622(97)00061-6. [DOI] [PubMed] [Google Scholar]
  15. Sirangelo I., Bismuto E., Tavassi S., Irace G. Apomyoglobin folding intermediates characterized by the hydrophobic fluorescent probe 8-anilino-1-naphthalene sulfonate. Biochim Biophys Acta. 1998 Jun 11;1385(1):69–77. doi: 10.1016/s0167-4838(98)00038-7. [DOI] [PubMed] [Google Scholar]
  16. Springer B. A., Sligar S. G. High-level expression of sperm whale myoglobin in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8961–8965. doi: 10.1073/pnas.84.24.8961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965 Sep;13(2):482–495. doi: 10.1016/s0022-2836(65)80111-5. [DOI] [PubMed] [Google Scholar]
  18. TEALE F. W. Cleavage of the haem-protein link by acid methylethylketone. Biochim Biophys Acta. 1959 Oct;35:543–543. doi: 10.1016/0006-3002(59)90407-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES