Fluorescence Lifetime Imaging in Scanning Microscopy

  • H. C. Gerritsen
  • A. Draaijer
  • D. J. van den Heuvel
  • A. V. Agronskaia


It was not until the 1930s that measurement of the fluorescence lifetime (τ f ) and the confirmation of theory on this phenomenon became possible (Pringsheim, 1961). Before that time only phosphorescence lifetimes had been measured, evidently because this phenomenon is one or more orders of magnitude slower.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Atherton, S.J., and Beaumont, P.C., 1984, Ethidium bromide as a fluorescent probe of the accessibility of water to the interior of DNA, Photobiochem Photobiophys 8:103–113.Google Scholar
  2. Ballew, R.M., and Demas, J.N., 1989, An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays, Anal. Chem. 61:30–33.Google Scholar
  3. Becker, W., Bergmann, A., Biskup, C., Kelbauskas, L., Zimmer, T., Klöcker, N., and Benndorf, K., 2003, High resolution TCSPC lifetime imaging, Proc. SPIE 4963:175–184.CrossRefGoogle Scholar
  4. Berlin, J., and Konishi, M., 1993, Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators, Biophys. J. 65:1632–1647.Google Scholar
  5. Bradshaw, D.J., Marsh, P.D., Gerritsen, H., Vroom, J., Watson, G.K., and Allison, C., 1998, Detection of pH gradients in biofilms using 2-photon excitation microscopy, J. Dent. Res. 77:2854.Google Scholar
  6. Bugiel, I., König, K., and Wabnitz, H., 1989, Investigation of cells by fluorescence laser scanning microscopy with subnanosecond time resolution, Laser Life Sci. 3:1–7.Google Scholar
  7. Buurman, E.P., Sanders, R., Draaijer, A., Gerritsen, H.C., Van Veen, J.J.F., Houpt, P.M., and Levine, Y.K., 1992, Fluorescence lifetime imaging using a confocal laser scanning microscope, Scanning 14:155–159.Google Scholar
  8. Clark, R.J.H., and Hester, R.E., 1989, Time Resolved Spectroscopy, John Wiley and Sons, Chichester, United Kingdom.Google Scholar
  9. Clays, K., Giambattista, M.D., Persoons, A., and Engelborghs, Y., 1991, A fluorescence lifetime study of virginiamycin S using multifrequency phase fluorometry, Biochemistry 30:7271–7276.CrossRefPubMedGoogle Scholar
  10. Clegg, R.M., 1996, Fluorescence resonance energy transfer, In: Fluorescence Imaging Spectroscopy and Microscopy (X.F. Wang and B. Herman, eds.), John Wiley and Sons, New York, pp. 179–236.Google Scholar
  11. de Grauw, C.J., and Gerritsen, H.C., 2001, Multiple time-gate module for fluorescence lifetime imaging, Appl. Spectrosc. 55:670–678.CrossRefGoogle Scholar
  12. Demas, J.N., 1983, Excited State Lifetime Measurements, Academic Press, New York.Google Scholar
  13. Dix, J.A., and Verkman, A.S., 1990, Mapping of fluorescence anisotropy in living cells by ratio imaging. Application to cytoplasmic viscosity, Biophys. J. 57:231–240.CrossRefPubMedGoogle Scholar
  14. Draaijer, A., and Houpt, P.M., 1988, A standard video-rate confocal laser scanning reflection and fluorescence microscope, Scanning 10:139–Google Scholar
  15. Duportail, G., Mauss, Y., and Chambron, J., 1977, Quantum yields and fluorescence lifetimes of acridine derivatives interacting with DNA, Biopolymers 16:1397–1413.CrossRefPubMedGoogle Scholar
  16. Förster, T., 1946, Energiewanderung und Fluoreszenz, Naturwissenschaften 6:166–175.CrossRefGoogle Scholar
  17. French, T., So, P.T.C., Dong, C.Y., Berland, K.M., and Gratton, E., 1998, Fluorescence lifetime imaging techniques for microscopy, Methods Cell Biol. 56:277–304.CrossRefPubMedGoogle Scholar
  18. Gadella, T.W., Jovin, T.M., and Clegg, R.M., 1993, Fluorescence lifetime imaging microscopy (FLIM) — spatial resolutions of microstructures on the nanosecond time scale, Biophys. Chem. 48:221–239.Google Scholar
  19. Gafni, A., and Brand, L., 1978, Excited state proton transfer reactions of acridine studied by nanosecond fluorometry, Chem. Phys. Lett. 58:346.CrossRefGoogle Scholar
  20. Gerritsen, H.C., Asselbergs, M.A.H., Agronskaia, A.V., and Van Sark, W., 2002, Fluorescence lifetime imaging in scanning microscopes: Acquisition speed, photon economy and lifetime resolution, J. Microsc. 206:218–224.CrossRefGoogle Scholar
  21. Gerritsen, H.C., Sanders, R., Draaijer, A., and Levine, Y.K., 1996, The photon economy of fluorescence lifetime imaging, Scanning 18:55–56.Google Scholar
  22. Gerritsen, H.C., Sanders, R., Draaijer, A., and Levine, Y.K., 1997, Fluorescence lifetime imaging of oxygen in living cells, J. Fluoresc. 7:11–16.CrossRefGoogle Scholar
  23. Ghiggino, K.P., Harris, M.R., and Spizzirri, P.G., 1992, Fluorescence lifetime measurements using a novel fiberoptic laser scanning confocal microscope, Rev. Sci. Instrum. 63:2999–3002.CrossRefGoogle Scholar
  24. Gratton, E., and Limkeman, M., 1983, Acontinuously variable frequency crosscorrelation phase fluorometer with picosecond resolution, Biophys. J. 44:315–324.Google Scholar
  25. Gratton, E., Breusegem, S., Sutin, J., Ruan, Q., and Barry, N., 2003, Fluorescence lifetime imaging for the two photon microscope: Time-domain and frequency-domain methods, J. Biomed. Opt. 8:381–390.CrossRefPubMedGoogle Scholar
  26. Gratton, E., Jameson, D.M., and Hall, R.D., 1984, Multifrequency phase and modulation fluorometry, Ann. Rev. Biophys. Bioeng. 13:105–124.CrossRefGoogle Scholar
  27. Grynkiewicz, G., Poenie, M., and Tsien, R.Y., 1985, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260:3440–3450.PubMedGoogle Scholar
  28. Hamamatsu, 1997. Data sheet MCP-PMT R3809U-50 Series.Google Scholar
  29. 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. 7:85–92.Google Scholar
  30. Jameson, D.M., and Gratton, E., 1983, Analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry, In: New Directions in Molecular Luminescence (D. Eastwood and L. Cline-Love, eds.), American Society for Testing and Materials, Philadelphia, pp. 67–81.Google Scholar
  31. Keating, S.M., Wensell, T.G., Meyer, T., and Stryer, L., 1989, Nanosecond fluorescence and emission anisotropy kinetics of fura-2 in single cells, Biophys. J. 55:518a.Google Scholar
  32. Kollner, M., and Wolfrum, J., 1992, How many photons are necessary for fluorescence-lifetime measurements? Chem. Phys. Lett. 200:199–204.CrossRefGoogle Scholar
  33. Krishnan, R.V., Saitoh, H., Terada, H., Centonze, V.E., and Herman, B., 2003, Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera, Rev. Sci. Instrum. 74:2714–2721.CrossRefGoogle Scholar
  34. Kwak, E.-S., and Vanden Bout, D.A., 2003, Fully time-resolved near-field scanning optical microscopy fluorescence imaging, Anal. Chim. Acta.Google Scholar
  35. 496:259–266.Google Scholar
  36. Lakowicz, J.R., 1983, Principles of Fluorescence Spectroscopy, Plenum Press, New York.Google Scholar
  37. Lakowicz, J.R., 1999, Principles of Fluorescence Spectroscopy, Plenum Press, New York.Google Scholar
  38. Lakowicz, J.R., and Berndt, K.W., 1991, Lifetime-selective fluorescence imaging using an Rf phase-sensitive camera, Rev. Sci. Instrum. 62: 1727–1734.CrossRefGoogle Scholar
  39. Lakowicz, J.R., and Szmacinski, H., 1993, Fluorescence lifetime-based sensing of Ph, Ca-2(+), K+ and glucose. Sens. Actuator B Chem. 11:133–143.CrossRefGoogle Scholar
  40. Lakowicz, J.R., Szmacinski, H., Lederer, W.J., Kirby, M.S., Johnson, M.L., and Nowaczyk, K., 1994, Fluorescence lifetime imaging of intracellular calcium in Cos cells using Quin-2, Cell Calcium 15:7–27.CrossRefPubMedGoogle Scholar
  41. Laws, W.R., and Brand, L., 1979, Analysis of two-state excited-state reactions. The fluorescence decay of 2-naphtol, J. Phys. Chem. 83:795–802.CrossRefGoogle Scholar
  42. Marriott, G., Clegg, R.M., Arndtjovin, D.J., and Jovin, T.M., 1991a, Time resolved imaging microscopy — phosphorescence and delayed fluorescence imaging, Biophys. J. 60:1374–1387.Google Scholar
  43. Marriott, G., Clegg, R.M., Arndt-Jovin, D.J., and Jovin, T.M., 1991b, Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging, Biophys. J. 60:1374–1387.CrossRefPubMedGoogle Scholar
  44. McLoskey, D., Birch, D.J.S., Sanderson, A., Suhling, K., Welch, E., and Hicks, P.J., 1996, Multiplexed single-photon counting. 1. A time-correlated fluorescence lifetime camera, Rev. Sci. Instrum. 67:2228–2237.CrossRefGoogle Scholar
  45. Minami, T., and Hirayama, S., 1990, High-quality fluorescence decay curves and lifetime imaging using an elliptic scan streak camera, J. Photochem. Photobiol. A Chem. 53:11–21.CrossRefGoogle Scholar
  46. Morgan, C.G., Mitchel, A.C., and Murray, J.G., 1992, Prospects for confocal imaging based on nanosecond fluorescence decay time, J. Microsc. 165:49–60.Google Scholar
  47. Morgan, C.G., Mitchell, A.C., and Murray, J.G., 1990, Nano-second timeresolved fluorescence microscopy: principles and practice, Trans. R. Microsc. Soc. 1:463–466.Google Scholar
  48. Ni, T.Q., and Melton, L.A., 1991, Fluorescence lifetime imaging — an approach for fuel equivalence ratio imaging, Appl. Spectrosc. 45:938–943.CrossRefGoogle Scholar
  49. O’Connor, D.V., and Phillips, D., 1984, Time-Correlated Single Photon Counting, Academic Press, London.Google Scholar
  50. Periasamy, A., Sharman, K.K., and Demas, J.N., 1999, Fluorescence lifetime imaging microscopy using rapid lifetime determination method: Theory and applications, Biophys. J. 76:A10.Google Scholar
  51. Periasamy, A., Wodnicki, P., Wang, X.F., Kwon, S., Gordon, G.W., and Herman, B., 1996, Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system, Rev. Sci. Instrum. 67:3722–3731.CrossRefGoogle Scholar
  52. Piston, D.W., Sandison, D.R., and Webb, W.W., 1992, Time-resolved fluorescence imaging and background rejection by two-photon excitation in laser scanning microscopy, SPIE 1640:379–387.CrossRefGoogle Scholar
  53. Pringsheim, P., 1961, Fluorescence and Phosphorescence, Interscience Publishers, New York.Google Scholar
  54. Rink, T.J., Tsien, R.Y., and Pozzan, T., 1982, Cytoplasmic pH and free Mg2+ in lymphocytes, J. Cell Biol. 95:189–196.CrossRefGoogle Scholar
  55. Sanders, R., Draaijer, A., Gerritsen, H.C., Houpt, P.M., and Levine, Y.K., 1995, Quantitative Ph imaging in cells using confocal fluorescence lifetime imaging microscopy, Anal. Biochem. 227:302–308.Google Scholar
  56. Sanders, R., Gerritsen, H., Draaijer, A., Houpt, P., and Levine, Y.K., 1994, Fluorescence lifetime imaging of free calcium in single cells, Bioimaging 2:131–138.CrossRefGoogle Scholar
  57. Sauer, M., Schulz, A., Seeger, S., and Wolfrum, J., 1993, Design of multiplex dyes, Phys. Chem. 17341737.Google Scholar
  58. Schneckenburger, H., and Konig, K., 1992, Fluorescence decay kinetics and imaging of Nad(P)H and flavins as metabolic indicators, Opt. Eng. 31:1447–1451.Google Scholar
  59. Scully, A.D., Ostler, R.B., Phillips, D., O’Neill, P.O., Townsend, K.M., Parker, A.W., and MacRobert, A.J., 1997, Application of fluorescence lifetime imaging microscopy to the investigation of intracellular PDT mechanisms, Bioimaging 5:9–18.CrossRefGoogle Scholar
  60. So, P.T.C., French, T., Yu, W.M., Berland, K.M., Dong, C.Y., and Gratton, E., 1996, Two photon microscopy: Time-resolved and intensity imaging, In: Fluorescence Imaging Spectroscopy and Microscopy (X.F. Wang and B. Herman, eds.), John Wiley and Sons, New York, pp. 351–371.Google Scholar
  61. Squire, A., Verveer, P.J., and Bastiaens, P.I.H., 2000, Multiple frequency fluorescence lifetime imaging microscopy, J. Microsc. 197:136–149.CrossRefGoogle Scholar
  62. Straughan, B.P., and Walker, S., 1976, Spectroscopy, Chapman and Hall, London.Google Scholar
  63. Sytsma, J., Vroom, J.M., De Grauw, C.J., and Gerritsen, H.C., 1998, Timegated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation, J. Microsc. 191:39–51.CrossRefGoogle Scholar
  64. Tsien, R.Y., and Poenie, M., 1986, Fluorescence ratio imaging: A new window into intracellular ionic signaling, Trends Biochem. Sci. 11:450–455.Google Scholar
  65. Van der Oord, C.J., De Grauw, C.J., and Gerritsen, H.C., 2001, Fluorescence lifetime imaging module LIMO for CLSM, Proc. SPIE 4252:119–123.CrossRefGoogle Scholar
  66. Vanderoord, C.J.R., Gerritsen, H.C., Rommerts, F.F.G., Shaw, D.A., Munro, I.H., and Levine, Y.K., 1995, Microvolume time-resolved fluorescence spectroscopy using a confocal synchrotron-radiation microscope, Appl. Spectrosc. 49:1469–1473.CrossRefGoogle Scholar
  67. Verveer, P.J., Squire, A., and Bastiaens, P.I.H., 2000, Global analysis of fluorescence lifetime imaging microscopy data, Biophys. J. 78:2127–Google Scholar
  68. Vroom, J.M., De Grauw, K.J., Gerritsen, H.C., Bradshaw, D.J., Marsh, P.D., Watson, G.K., Birmingham, J.J., and Allison, C., 1999, Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy, Appl. Environ. Microbiol. 65:3502–3511.PubMedGoogle Scholar
  69. Wang, X.F., Uchida, T., Coleman, D.M., and Minami, S., 1991, A 2- dimensional fluorescence lifetime imaging-system using a gated image intensifier, Appl. Spectrosc. 45:360–366.CrossRefGoogle Scholar
  70. Webb, S.E.D., Gu, Y., Leveque-Fort, S., Siegel, J., Cole, M.J., Dowling, K., Jones, R., French, P.M.W., Neil, M.A.A., Juskaitis, R., Sucharov, L.O.D., Wilson, T., and Lever, M.J., 2002, A widefield time-domain fluorescence lifetime imaging microscope with optical sectioning, Rev. Sci. Instrum. 73:1898–1907.CrossRefGoogle Scholar
  71. Whitaker, J.E., Haugland, R.P., and Prendergast, F.G., 1991, Spectral and photophysical studies of benzo[c]xanthene dyes: Dual emission pH sensors, Anal. Biochem. 194:330–344.Google Scholar
  72. Wouters, F.S., and Bastiaens, P.I.H., 1999, Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells, Curr. Biol. 9:1127–1130.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • H. C. Gerritsen
    • 1
  • A. Draaijer
    • 2
  • D. J. van den Heuvel
    • 1
  • A. V. Agronskaia
    • 1
  1. 1.Utrecht UniversityUtrechtThe Netherlands
  2. 2.TNO-VoedingUtrechtThe Netherlands

Personalised recommendations