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. 2002 Sep;83(3):1631–1649. doi: 10.1016/S0006-3495(02)73932-5

Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM).

Andrew H A Clayton 1, Quentin S Hanley 1, Donna J Arndt-Jovin 1, Vinod Subramaniam 1, Thomas M Jovin 1
PMCID: PMC1302260  PMID: 12202387

Abstract

We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and DC) between the polarized components of the fluorescence signal are recorded, leading to estimations of rotational correlation times and limiting anisotropies. Theory is developed that allows all the parameters of the hindered rotator model to be extracted from measurements carried out at a single modulation frequency. Two-dimensional image detection with a sensitive CCD camera provides wide-field imaging of dynamic depolarization with parallel interrogation of different compartments of a complex biological structure such as a cell. The concepts and technique of rFLIM are illustrated with a fluorophore-solvent (fluorescein-glycerol) system as a model for isotropic rotational dynamics and with bacteria expressing enhanced green fluorescent protein (EGFP) exhibiting depolarization due to homotransfer of electronic excitation energy (emFRET). The frequency-domain formalism was extended to cover the phenomenon of emFRET and yielded data consistent with a concentration depolarization mechanism resulting from the high intracellular concentration of EGFP. These investigations establish rFLIM as a powerful tool for cellular imaging based on rotational dynamics and molecular proximity.

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Selected References

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  1. Axelrod D. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys J. 1979 Jun;26(3):557–573. doi: 10.1016/S0006-3495(79)85271-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axelrod D. Fluorescence polarization microscopy. Methods Cell Biol. 1989;30:333–352. [PubMed] [Google Scholar]
  3. Bene L., Fulwyler M. J., Damjanovich S. Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements. Cytometry. 2000 Aug 1;40(4):292–306. [PubMed] [Google Scholar]
  4. Blackman S. M., Piston D. W., Beth A. H. Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer. Biophys J. 1998 Aug;75(2):1117–1130. doi: 10.1016/S0006-3495(98)77601-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buehler C., Dong C. Y., So P. T., French T., Gratton E. Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy. Biophys J. 2000 Jul;79(1):536–549. doi: 10.1016/S0006-3495(00)76315-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang M. C., Cross A. J., Fleming G. R. Internal dynamics and overall motion of lysozyme studied by fluorescence depolarization of the eosin lysozyme complex. J Biomol Struct Dyn. 1983 Oct;1(1):299–318. doi: 10.1080/07391102.1983.10507441. [DOI] [PubMed] [Google Scholar]
  7. Clore G. M., Driscoll P. C., Wingfield P. T., Gronenborn A. M. Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry. 1990 Aug 14;29(32):7387–7401. doi: 10.1021/bi00484a006. [DOI] [PubMed] [Google Scholar]
  8. Cross A. J., Fleming G. R. Influence of inhibitor binding on the internal motions of lysozyme. Biophys J. 1986 Sep;50(3):507–512. doi: 10.1016/S0006-3495(86)83488-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dix J. A., Verkman A. S. Mapping of fluorescence anisotropy in living cells by ratio imaging. Application to cytoplasmic viscosity. Biophys J. 1990 Feb;57(2):231–240. doi: 10.1016/S0006-3495(90)82526-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Florine-Casteel K. Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy. Biophys J. 1990 Jun;57(6):1199–1215. doi: 10.1016/S0006-3495(90)82639-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fushimi K., Dix J. A., Verkman A. S. Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging. Biophys J. 1990 Feb;57(2):241–254. doi: 10.1016/S0006-3495(90)82527-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gangal M., Cox S., Lew J., Clifford T., Garrod S. M., Aschbaher M., Taylor S. S., Johnson D. A. Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations. Biochemistry. 1998 Sep 29;37(39):13728–13735. doi: 10.1021/bi980560z. [DOI] [PubMed] [Google Scholar]
  13. Gautier I., Tramier M., Durieux C., Coppey J., Pansu R. B., Nicolas J. C., Kemnitz K., Coppey-Moisan M. Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. Biophys J. 2001 Jun;80(6):3000–3008. doi: 10.1016/S0006-3495(01)76265-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanley Q. S., Subramaniam V., Arndt-Jovin D. J., Jovin T. M. Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression. Cytometry. 2001 Apr 1;43(4):248–260. doi: 10.1002/1097-0320(20010401)43:4<248::aid-cyto1057>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]
  15. Jakobs S., Subramaniam V., Schönle A., Jovin T. M., Hell S. W. EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Lett. 2000 Aug 18;479(3):131–135. doi: 10.1016/s0014-5793(00)01896-2. [DOI] [PubMed] [Google Scholar]
  16. Jameson D. M., Sawyer W. H. Fluorescence anisotropy applied to biomolecular interactions. Methods Enzymol. 1995;246:283–300. doi: 10.1016/0076-6879(95)46014-4. [DOI] [PubMed] [Google Scholar]
  17. Kinosita K., Jr, Ikegami A., Kawato S. On the wobbling-in-cone analysis of fluorescence anisotropy decay. Biophys J. 1982 Feb;37(2):461–464. doi: 10.1016/S0006-3495(82)84692-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lakowicz J. R., Balter A. Theory of phase-modulation fluorescence spectroscopy for excited-state processes. Biophys Chem. 1982 Oct;16(2):99–115. doi: 10.1016/0301-4622(82)85012-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lakowicz J. R., Prendergast F. G., Hogen D. Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations. Biochemistry. 1979 Feb 6;18(3):508–519. doi: 10.1021/bi00570a021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lakowicz J. R., Szmacinski H., Nowaczyk K., Berndt K. W., Johnson M. Fluorescence lifetime imaging. Anal Biochem. 1992 May 1;202(2):316–330. doi: 10.1016/0003-2697(92)90112-k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lipari G., Szabo A. Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes. Biophys J. 1980 Jun;30(3):489–506. doi: 10.1016/S0006-3495(80)85109-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marriott G., Clegg R. M., Arndt-Jovin D. J., Jovin T. M. Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging. Biophys J. 1991 Dec;60(6):1374–1387. doi: 10.1016/S0006-3495(91)82175-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miyawaki A., Tsien R. Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 2000;327:472–500. doi: 10.1016/s0076-6879(00)27297-2. [DOI] [PubMed] [Google Scholar]
  24. Partikian A., Olveczky B., Swaminathan R., Li Y., Verkman A. S. Rapid diffusion of green fluorescent protein in the mitochondrial matrix. J Cell Biol. 1998 Feb 23;140(4):821–829. doi: 10.1083/jcb.140.4.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Patterson G. H., Knobel S. M., Sharif W. D., Kain S. R., Piston D. W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J. 1997 Nov;73(5):2782–2790. doi: 10.1016/S0006-3495(97)78307-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Patterson G. H., Piston D. W., Barisas B. G. Förster distances between green fluorescent protein pairs. Anal Biochem. 2000 Sep 10;284(2):438–440. doi: 10.1006/abio.2000.4708. [DOI] [PubMed] [Google Scholar]
  27. Periasamy N., Verkman A. S. Subtraction of background fluorescence in multiharmonic frequency-domain fluorimetry. Anal Biochem. 1992 Feb 14;201(1):107–113. doi: 10.1016/0003-2697(92)90181-6. [DOI] [PubMed] [Google Scholar]
  28. Squire A., Verveer P. J., Bastiaens P. I. Multiple frequency fluorescence lifetime imaging microscopy. J Microsc. 2000 Feb;197(Pt 2):136–149. doi: 10.1046/j.1365-2818.2000.00651.x. [DOI] [PubMed] [Google Scholar]
  29. Szmacinski H., Jayaweera R., Cherek H., Lakowicz J. R. Demonstration of an associated anisotropy decay by frequency-domain fluorometry. Biophys Chem. 1987 Sep;27(3):233–241. doi: 10.1016/0301-4622(87)80062-5. [DOI] [PubMed] [Google Scholar]
  30. Thevenin B. J., Periasamy N., Shohet S. B., Verkman A. S. Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1741–1745. doi: 10.1073/pnas.91.5.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tramier M., Kemnitz K., Durieux C., Coppey J., Denjean P., Pansu R. B., Coppey-Moisan M. Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells. Biophys J. 2000 May;78(5):2614–2627. doi: 10.1016/S0006-3495(00)76806-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Varma R., Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature. 1998 Aug 20;394(6695):798–801. doi: 10.1038/29563. [DOI] [PubMed] [Google Scholar]
  33. Vergani B., Kintrup M., Hillen W., Lami H., Piémont E., Bombarda E., Alberti P., Doglia S. M., Chabbert M. Backbone dynamics of Tet repressor alpha8intersectionalpha9 loop. Biochemistry. 2000 Mar 14;39(10):2759–2768. doi: 10.1021/bi9912591. [DOI] [PubMed] [Google Scholar]
  34. Verkman A. S., Armijo M., Fushimi K. Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains. Biophys Chem. 1991 Apr;40(1):117–125. doi: 10.1016/0301-4622(91)85036-p. [DOI] [PubMed] [Google Scholar]
  35. Verveer P. J., Squire A., Bastiaens P. I. Global analysis of fluorescence lifetime imaging microscopy data. Biophys J. 2000 Apr;78(4):2127–2137. doi: 10.1016/S0006-3495(00)76759-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Volkmer A., Subramaniam V., Birch D. J., Jovin T. M. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys J. 2000 Mar;78(3):1589–1598. doi: 10.1016/S0006-3495(00)76711-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

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