Phase-Sensitive and Phase-Resolved Emission Spectra

  • Joseph R. Lakowicz


In Chapter 5 we described the use of the FD method to measure lifetimes and to resolve complex intensity decays. In FD fluorometers, the sample is excited with intensity-modulated light, and one measures the phase shift and modulation of the emission, both relative to the excitation. The FD method also allows several other types of measurement which can be useful in special circumstances. One method is measurement of phase-sensitive intensities and/or emission spectra. Another method is to use the measured phase and modulation values to resolve the components of species in a mixture based on known decay times.


Emission Spectrum Phase Angle Image Intensifier Quinine Sulfate Fluorescence Lifetime Imaging Microscopy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Lakowicz, J. R., and Cherek, H., 1981, Phase-sensitive fluorescence spectroscopy. A new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores, J. Biochem. Biophys. Methods 5: 19–35.Google Scholar
  2. 2.
    Lakowicz, J. R., and Cherek, H., 1981, Resolution of heterogeneous fluorescence from proteins and aromatic amino acids by phase-sensitive detection of fluorescence, J. Biot. Chem. 256: 6348–6353.Google Scholar
  3. 3.
    Lakowicz, J. R., and Cherek, H., 1982, Resolution of heterogeneous fluorescence by phase-sensitive fluorescence spectroscopy, Biophys. J. 37: 148–150.CrossRefGoogle Scholar
  4. 4.
    Jameson, D. M., Gratton, E., and Hall, R. D., 1984, The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry, AppL Spectrosc. Rev. 20 (1): 55–106.CrossRefGoogle Scholar
  5. 5.
    McGown, L., and Bright, E, 1984, Phase-resolved fluorescence spectroscopy, Anal. Chem. 56: 1400–1415.Google Scholar
  6. 6.
    Fugate, R. D., Bartlett, J. D., and Mattheis, J. R., 1984, Phase-resolution in spectrofluorometric measurements: Applications to biochemical systems, BioTechniques 1984 (May/June): 174–180.Google Scholar
  7. 7.
    McGown, L. B., 1985, Determination of fluorescence lifetimes and heterogeneity analysis using time-independent phase-resolved intensity measurements, Anal. Instrum. 14 (3and4): 251–265.CrossRefGoogle Scholar
  8. 8.
    McGown, L. B., and Nithipatikom, K., 1991, Multicomponent determinations of polycyclic aromatic hydrocarbons using synchronous excitation phase-resolved fluorometry, in Advances in Multidimensional Luminescence, Vol. 1, I. M. Warner and L. B. McGown (eds.), JAI Press, Greenwich, Connecticut, pp. 97–109.Google Scholar
  9. 9.
    McGown, L. B., and Bright, F. V., 1987, Phase-resolved fluorescence in chemical analysis, Anal. Chem. 18: 245–298.Google Scholar
  10. 10.
    Demas, J. N., and Keller, R. A., 1985, Enhancement of luminescence and Raman spectroscopy by phase-resolved background suppression, Anal. Chem. 57: 538–545.CrossRefGoogle Scholar
  11. 11.
    Nithipatikom, K., and McGown, L. B., 1987, Phase-resolved suppression of scattered light in total luminescence spectra, Appl. Spectrosc. 41: 1080–1082.CrossRefGoogle Scholar
  12. 12.
    Wirth, M. J., and Chou, S.-H., 1988, Comparison of time and frequency domain methods for rejecting fluorescence from Raman spectra, Anal. Chem. 60: 1882–1886.CrossRefGoogle Scholar
  13. 13.
    Nithipatikom, K., and McGown, L. B., 1986, Elimination of scatter background in synchronous excitation spectrofluorometry by the use of phase-resolved fluorescence spectroscopy, AnaL Chem. 58: 31453147.Google Scholar
  14. 14.
    Lakowicz, J. R., and Baiter, A., 1982, Direct recording of the initially excited and the solvent relaxed fluorescence emission spectra of tryptophan by phase sensitive detection of fluorescence, Photochem. Photobiol. 36: 125–132.CrossRefGoogle Scholar
  15. 15.
    Lakowicz, J. R., and Batter, A., 1982, Detection of the reversibility of an excited-state reaction by phase-modulation fluorometry, Chem. Phys. Lett. 92 (2): 117–121.CrossRefGoogle Scholar
  16. 16.
    Lakowicz, J. R., Thompson, R. B., and Cherek, H., 1983, Phase fluorometric studies of spectral relaxation at the lipid—water interface of phospholipid vesicles, Biochim. Biophys. Acta 734: 295–308.Google Scholar
  17. 17.
    Nithipatikom, K., and McCown, L. B., 1987, Five-and six-component determinations using phase-resolved fluorescence spectroscopy and synchronous excitation, AppL Spectrosc. 41: 395–398.CrossRefGoogle Scholar
  18. 18.
    Bright, F. V., and McGown, L. B., 1986, Three-component determinations using fluorescence anisotropy measurements and wavelength selectivity, Anal. Chem. 58: 1424–1427.CrossRefGoogle Scholar
  19. 19.
    Bright, F. V., and McGown, L. B., 1985, Phase-resolved fluorometric determinations of four-component systems using two modulation frequencies, Anal. Chem. 57: 2877–2880.Google Scholar
  20. 20.
    Bright, F. V., and McGown, L. B., 1985, Four-component determinations using phase-resolved fluorescence spectroscopy, Anal. Chem. 57: 55–59.Google Scholar
  21. 21.
    Vitense, K. R., and McGown, L. B., 1987, Simultaneous determination of metals in two-component mixtures with 5-sulfo-8-quinolinol by using phase-resolved fluorimetry, Anal. Chim. Acta 193: 119125.Google Scholar
  22. 22.
    Nithipatikom, K., and McGown, L. B., 1986, Multidimensional data formats for phase-resolved fluorometric multicomponent determinations using synchronous excitation and emission spectra, Anal. Chem. 58: 2469–2473.CrossRefGoogle Scholar
  23. 23.
    Bright, F. V., and McGown, L. B., 1984, Elimination of bilirubin interference in fluorometric determination of fluorescein by phase-resolved fluorescence spectrometry, Anal. Chim. Acta 162: 275–283.CrossRefGoogle Scholar
  24. 24.
    Lakowicz, J. R., and Keating, S., 1983, Binding of an indole derivative to micelles as quantified by phase-sensitive detection of fluorescence, J. Biol. Chem. 258: 5519–5524.Google Scholar
  25. 25.
    McGown, L. B., 1984, Phase-resolved fluoroimetric determination of two albumin-bound fluorescein species, Anal. Chim. Acta 157: 327–332.CrossRefGoogle Scholar
  26. 26.
    Nithipatikom, K., and McGown, L. B., 1989, Studies of the homogeneous immunochemical determination of insulin by using a fluorescent label, Talanta 36 (112): 305–309.CrossRefGoogle Scholar
  27. 27.
    Nithipatikom, K., and McGown, L. B., 1987, Homogeneous immunochemical technique for determination of human lactoferrin using excitation transfer and phase-resolved fluorometry, Anal. Chem. 59: 423–427.CrossRefGoogle Scholar
  28. 28.
    Tahboub, Y. R., and McGown, L. B., 1986, Phase-resolved fluoroimmunoassay of human serum albumin, Anal. Chim. Acta 182: 185191.Google Scholar
  29. 29.
    Veselova, T. V., Cherkasov, A. S., and Shirokov, V. I., 1970, Fluorometric method for individual recording of spectra in systems containing two types of luminescent centers, Opt. Spectrosc. 29: 617–618.Google Scholar
  30. 30.
    Grafton, E., and Jameson, D. M., 1985, New approach to phase and modulation resolved spectra, Anal. Chem. 57: 1694–1697.CrossRefGoogle Scholar
  31. 31.
    Lakowicz, J. R., and Baiter, A., 1982, Theory of phase-modulation fluorescence spectroscopy for excited state processes, Biophys. Chem. 16: 99–115.CrossRefGoogle Scholar
  32. 32.
    Lakowicz, J. R., and Baiter, A., 1982, Analysis of excited-state processes by phase-modulation fluorescence spectroscopy, Biophys. Chem. 16: 117–132.CrossRefGoogle Scholar
  33. 33.
    Veselova, T. V., Limareva, L. A., Cherkasov, A. S., and Shirokov, V. I., 1965, Fluorometric study of the effect of solvent on the fluorescence spectrum of 3-amino-N-methylphthalimide, Opt. Spectrosc. 19: 39–43.Google Scholar
  34. 35.
    Barcellona, M. L., and Gratton, E., 1990, The fluorescence properties of a DNA probe, Eur. Biophys. J. 17: 315–323.CrossRefGoogle Scholar
  35. 36.
    Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., and Johnson, M. L., 1992, Fluorescence lifetime imaging of free and protein-bound NADH, Proc. Natl. Acad. Sci. U.S.A. 89: 1271–1275.CrossRefGoogle Scholar
  36. 37.
    Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., Berndt, B. W., and Johnson, M. L., 1992, Fluorescence lifetime imaging, Anal. Biochem. 202: 316–330.CrossRefGoogle Scholar
  37. 38.
    Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., and Johnson, M. L., 1992, Fluorescence lifetime imaging of calcium using quin-2, Cell Calcium 13:131–147.Google Scholar
  38. 39.
    Szmacinski, H., Lakowicz, J. R., and Johnson, M. L., 1994, Fluorescence lifetime imaging microscopy: Homodyne technique using high-speed gated image intensifier, Methods Enzymol. 240: 723748.Google Scholar
  39. 40.
    Draaijer, A., Sanders, R., and Gerritsen, H. C., 1995, Fluorescence lifetime imaging, a new tool in confocal microscopy, in Handbook of Biological Confocal Microscopy, J. B. Pawley (ed.), Plenum Press, New York, pp. 491–505.Google Scholar
  40. 41.
    French, T., Gratton, E., and Maier, J., 1992, Frequency domain imaging of thick tissues using a CCD, Proc. SPIE 1640: 254–261.CrossRefGoogle Scholar
  41. 42.
    Lakowicz, J. R., and Szmacinski, H., 1996, Imaging applications of time-resolved fluorescence spectroscopy, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman (eds.), John Wiley and Sons, New York, pp. 273–311.Google Scholar
  42. 43.
    Wang, X. F., Periasamy, A., Wodnicki, P., Gordon, G. W., and Herman, B., 1996, Time-resolved fluorescence lifetime imaging microscopy: Instrumentation and biomedical applications, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman (eds.), John Wiley and Sons, New York, pp. 313–350.Google Scholar
  43. 44.
    Gerritsen, H., and Draaijer, A., 1997, Second International Lifetime Imaging Meeting, Utrecht, The Netherlands, June 14, 1996, J. Fluoresc. 7 (1): 1–98.CrossRefGoogle Scholar
  44. 45.
    Herman, B., Wodnicki, P., Kwon, S., Periasamy, A., Gordon, G. W., Mahajan, N., and Wang, X. F. 1997, Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy, J. Fluoresc. 7(1):85–91.Google Scholar
  45. 46.
    Wagnieres, G., Mizeret, J., Studzinski, A., and van den Bergh, H., 1997, Frequency-domain fluorescence lifetime imaging for endoscopie clinical cancer photodetection: Apparatus design and preliminary results, J. Fluoresc. 7 (1): 75–83.CrossRefGoogle Scholar
  46. 47.
    Hartmann, P., and Ziegler, W., 1996, Lifetime imaging of luminescent oxygen sensors based on all-solid-state technology, Anal. Chem. 58: 4512–4514.CrossRefGoogle Scholar
  47. 48.
    Cubeddu, R., Canti, G., Pifferi, A., Taroni, E, and Valentini, G.,1997, Fluorescence lifetime imaging of experimental tumors in hematoporphyrin derivative-sensitized mice, Photochem. Photobiol. 66: 229–236.Google Scholar
  48. 49.
    Morgan, C. G., and Mitchell, A. C., 1996, Fluorescence lifetime imaging: An emerging technique in fluorescence microscopy, Chromosome Res. 4: 261–263.CrossRefGoogle Scholar
  49. 50.
    Schneider, P. C., and Clegg, R. M., 1997, Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications, Rev. Sci. Instrum. 68: 4107–4119.CrossRefGoogle Scholar
  50. 51.
    Dowling, K., Hyde, S. C. W., Dainty, J. C., French, P. M. W., and Hares, J. D., 1997, 2-D fluorescence lifetime imaging using a time-gated image intensifier, Opt. Commun. 135: 27–31.Google Scholar
  51. 52.
    Gadella, T. W. J., van Hoek, A., and Visser, A. J. W. G., 1997, Construction and characterization of a frequency-domain fluorescence lifetime imaging microscopy system, J. Fluoresc. 7 (1): 35–43.CrossRefGoogle Scholar
  52. 53.
    vandeVen, M., and Gratton, E., 1993, Time-resolved fluorescence lifetime imaging, in Optical Microscopy: Emerging Methods and Applications, B. Herman and J. J. Lemasters (eds.), Academic Press, New York, pp. 373–402.Google Scholar
  53. 54.
    Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., Lederer, W. J., Kirby, M. S., and Johnson, M. L., 1994, Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2, Cell Calcium 15: 7–27.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1999

Authors and Affiliations

  • Joseph R. Lakowicz
    • 1
  1. 1.University of Maryland School of MedicineBaltimoreUSA

Personalised recommendations