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Pitfalls and Their Remedies in Time-Resolved Fluorescence Spectroscopy and Microscopy

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Abstract

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of time- and frequency-domain equipment as well as practical remedies against common error conditions, elucidated with several graphs to aid the researcher in visual inspection and quality-control of collected data. A range of artifacts due to sample preparation or to optical and electronic pitfalls are discussed, as are remedies against them. Also recommended data analysis strategies are described.

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References

  1. E. Gaviola (1926). Ein Fluorimeter: Apparat zur Messung von Fluorescenzabklingungszeiten. Z. Physik 42, 853–861; E. Gaviola (1926). Die Abklingungszeiten der Fluorescenz von Farbstoffl¨sungen. Ann. Physik 81, 681–710.

    Google Scholar 

  2. B. Valeur (2002). Molecular Fluorescence. Principles and Applications, Wiley-VCH, Weinheim.

    Google Scholar 

  3. E. Gratton, D. M. Jameson, N. Rosato, and G. Weber (1984). Multifrequency cross-correlation phase fluorometer using synchrotron radiation. Rev. Sci. Instrum. 55, 486–494.

    Google Scholar 

  4. S. Landgraf (2001). Application of semiconductor light sources for investigations of photochemical reactions. Spectrochimica Acta A 57, 2029–2048; S. Landgraf and G. Grampp (1998). Application of laser diodes and ultrabright light emitting diodes for the determination of fluorescence lifetimes in the nano- and subnanosecond region. J. Inform. Record. 24, 141–148.

    Google Scholar 

  5. U. K. Tirlapur and K. K¨nig (2002). Two-photon near-infrared femtosecond laser scanning confocal microscopy in plant biology. in A. Diaspro (Ed.), Confocal and Two-Photon Microscopy. Foundations, Applications, and Advances, Wiley-Liss, New York, pp. 449–468.

    Google Scholar 

  6. T. W. J. Gadella (1999). Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and applications. In W. T. Mason (Ed.), Fluorescent and Luminescent Probes for Biological Activity: A practical Guide to Technology for Quantitative Real-Time Analysis, Academic Press, San Diego, pp. 467–479.

    Google Scholar 

  7. D. Axelrod, E. H. Hellen, and R. M. Fulbright (1992). Total internal reflection fluorescence. In J. R. Lakowicz (Ed.), Topics in Fluorescence Spectroscopy: Biochemical Applications, Plenum, New York, pp. 289–343.

    Google Scholar 

  8. V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman (2003). Fluorescence resonance energy transfer imaging microscopy. In G. Marriott and I. Parker (Eds.), Methods in Enzymology. Biophotonics, Part A, Academic Press, Amsterdam, pp. 542–560.

    Google Scholar 

  9. J. R. Lakowicz (1999). Principles of Fluorescence Spectroscopy, Plenum, New York.

    Google Scholar 

  10. D. V. O’Connor and D. Phillips (1984). Time-correlated single photon counting, Academic Press, London.

    Google Scholar 

  11. N. Boens (1991). Pulse fluorometry. In W. R. G. Baeyens, D. De Keukeleire, and K. Korkidis (Eds.), Luminescence Techniques in Chemical and Biochemical Analysis, Marcel Dekker, New York, pp. 21–45.

    Google Scholar 

  12. E. Gratton, D. M. Jameson, and R. D. Hall (1984). Multifrequency phase and modulation fluorometry. Ann. Rev. Biophys. Bioeng. 13, 105–124.

    Google Scholar 

  13. B. Valeur (2004). Pulse and phase fluorometries. An objective comparison. In M. Hof, R. Hutterer, and V. Fidler (Eds.), Fluorescence Spectroscopy in Biology. Advanced Methods and Their Applications to Membranes, Proteins, DNA, and Cells, Springer-Verlag, pp. 30–48.

  14. E. Gratton, S. Breusegem, J. Sutin, and Q. Q. Ruan (2003). Fluorescence lifetime imaging for the two-photon microscope: Time-domain and frequency-domain methods. J. Biomed. Optics 8, 381–390.

    Google Scholar 

  15. M. vandeVen and E. Gratton (1993). Time-resolved fluorescence lifetime imaging. In B. Herman and J. J. Lemasters (Eds.), Optical Microscopy. Emerging Methods and Applications, Academic Press, San Diego, pp. 373–402.

    Google Scholar 

  16. R. M. Clegg, O. Holub, and C. Gohlke (2003). Fluorescence lifetime-resolved imaging: Measuring lifetimes in an image. Biophotonics A 360, 509–542.

    Google Scholar 

  17. P. J. Verveer, S. Squier, and P. I. H. Bastiaens (2001). Frequency-domain fluorescence lifetime imaging Microscopy: A window on the biochemical landscape of the cell. In A. Periasamy (Ed.), Methods in Cellular Imaging, Oxford University Press, Oxford, pp. 273–294.

    Google Scholar 

  18. R. B. Thompson and E. Gratton (1988). Phase fluorometric method for determination of standard lifetimes. Anal. Chem. 60, 670–674.

    Google Scholar 

  19. J. N. Demas (1983). Excited State Lifetime Measurements, Academic Press, New York.

    Google Scholar 

  20. G. R. Holtom (1990). Artifacts and diagnostics in fast fluorescence measurements. In J. R. Lakowicz (Ed.), Time-Resolved Laser Spectroscopy in Biochemistry II. SPIE Proceedings, Vol. 1204, Los Angeles, pp. 2–12.

  21. R. Kay (2004). Detecting and minimizing zinc contamination in physiological solutions. BMC Physiol. 4, 4–9.

    Google Scholar 

  22. E. Bucci, H. Malak, C. Fronticelli, I. Gryczynski, G. Laczko, and J. R. Lakowicz (1988). Time-resolved emission spectra of hemoglobin on the picosecond time scale. Biophys. Chem. 32, 187–198.

    Google Scholar 

  23. B. W. Van der Meer (1988). Biomembrane structure and dynamics by fluorescence. In H. J. Hilderson (Ed.), Fluorescence Studies on Biological Membranes. Vol. 13: Subcellular Biochemistry, Plenum, New York, pp. 1–53.

    Google Scholar 

  24. L. A. Bagatolli and E. Gratton (2001). Direct observation of lipid domains in free-standing bilayers using two-photon excitation fluorescence microscopy. J. Fluoresc. 11, 141–160.

    Google Scholar 

  25. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin (2002). Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM). Biophys. J. 83, 1631–1649.

    Google Scholar 

  26. K. K¨nig (2001). Cellular response to laser radiation in fluorescence microscopes. In A. Periasamy (Ed.), Methods in Cellular Imaging, Oxford University Press, Oxford, pp. 236–251.

    Google Scholar 

  27. K. K¨nig and U. K. Tirlapur (2002). Cellular and subcellular perturbations during multiphoton microscopy. In A. Diaspro (Ed.), Confocal and Two-Photon Microscopy. Foundations, Applications and Advances, Wiley-Liss, New York, pp. 191–205.

    Google Scholar 

  28. E. M. Gill, G. M. Palmer, and N. Ramanujam (2003). Steady-state fluorescence imaging of Neoplasia. In G. Marriott and I. Parker (Eds.), Methods in Enzymology. Biophotonics, Part B, Academic Press, Amsterdam, pp. 452–481.

    Google Scholar 

  29. P. Herman, B. P. Maliwal, H. J. Lin, and J. R. Lakowicz. (2001). Frequency-domain fluorescence microscopy with the LED as a light source. J. Microsc. 203, 176–181.

    Google Scholar 

  30. T. C. W. Brelje, M. W. Wessendorf, and R. L. Sorenson (1993). Multicolor laser scanning confocal immunofluorescence microscopy: Practical applications and limitations. In B. Matsumoto (Ed.), Cell Biological Applications of Confocal Microscopy, Academic Press, San Diego, pp. 97–181.

    Google Scholar 

  31. E. Gratton and M. J. vande Ven (1995). Laser sources for confocal microscopy. In J. B. Pawley (Ed.), Handbook of Biological Confocal Microscopy, Plenum, New York, pp. 69–97.

    Google Scholar 

  32. R. Wolleschensky, M. E. Dickinson, and S. E. Fraser (2002). Group-velocity dispersion and fiber delivery in multiphoton laser scanning microscopy. In A. Diaspro (Ed.), Confocal and Two-Photon Microscopy. Foundations, Applications and Advances, Wiley-Liss, New York, pp. 171–190.

    Google Scholar 

  33. E. Gratton and M. Limkeman (1983). A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys. J. 44, 315–324.

    Google Scholar 

  34. D. M. Jameson, C. J. C. Croney, and P. D. J. Moens (2003). Fluorescence: Basic concepts, practical aspects, and some anecdotes. In G. Marriott and I. Parker (Eds.), Methods in Enzymology. Biophotonics Part A, Academic Press, Amsterdam, pp. 1–43.

    Google Scholar 

  35. D. Bebelaar (1986). Compensator for the time dispersion in a monochromator. Rev. Sci. Instrum. 57, 1686–1687.

    Google Scholar 

  36. D. A. Long (1977). Raman Spectroscopy, McGraw-Hill, New York.

    Google Scholar 

  37. J. R. Lakowicz, G. Laczko, and I. Gryczynski (1986). 2-GHz frequency-domain fluorometer. Rev. Sci. Instrum. 57, 2499– 2506.

    Google Scholar 

  38. K. W. Berndt and J. R. Lakowicz (1990). 4-GHz Internal MCP-photomultiplier cross-correlation. Rev. Sci. Instrum. 61, 2557– 2565.

    Google Scholar 

  39. G. Laczko, I. Gryczynski, Z. Gryczynski, W. Wiczk, H. Malak, and J. R. Lakowicz (1990). A 10-GHz frequency-domain fluorometer. Rev. Sci. Instrum. 61, 2331–2337.

    Google Scholar 

  40. E. Gratton and M. vandeVen (1990). A superheterodyning microwave frequency-domain fluorometer. Biophys. J. 57, A378.

    Google Scholar 

  41. B. Barbieri, F. De Piccoli, M. vande Ven, and E. Gratton (1990). What determines the uncertainty of phase and modulation measurements in frequency domain fluorometry. In J. R. Lakowicz (Ed.), Time-Resolved Laser Spectroscopy in Biochemistry II. SPIE Proceedings, Vol. 1204, Los Angeles, pp. 158–170.

  42. M. A. Franceschini, S. Fantini, and E. Gratton (1994). LEDs in frequency-domain spectroscopy of tissues. in Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases. SPIE Proceedings, Vol. 2135, Los Angeles, pp. 300– 306.

  43. B. Barbieri, F. De Piccoli, and E. Gratton (1989). Synthesizers phase noise in frequency-domain fluorometry. Rev. Sci. Instrum. 60, 3201–3206.

    Google Scholar 

  44. D. W. Piston, G. Marriott, T. Radivoyevich, R. M. Clegg, T. M. Jovin, and E. Gratton. (1989). Wide-band acousto-optic light modulator for frequency domain fluorometry and phosphorimetry. Rev. Sci. Instrum. 60, 2596–2600

    Google Scholar 

  45. E. Gratton, B. Feddersen, and M. vande Ven (1990). Parallel acquisition of fluorescence decay using array detectors. In J. R. Lakowicz (Ed.), Time-Resolved Laser Spectroscopy in Biochemistry II. SPIE Proceedings, Vol. 1204, Los Angeles, pp. 21–25.

  46. S. Canonica, J. Forrer, and U. P. Wild (1985). Improved timing resolution using small side-on photomultipliers in single photon counting. Rev. Sci. Instrum. 56, 1754–1758.

    Google Scholar 

  47. M. P. Gordon and P. R. Selvin (2003). A microcontroller-based failsafe for single photon counting modules. Rev. Sci. Instrum. 74, 1150–152.

    Google Scholar 

  48. M. vandeVen and E. Gratton (1993). Time-resolved fluorescence lifetime imaging. In B. Herman and J. J. Lemasters (Ed.), Optical Microscopy. Emerging Methods and Applications, Academic Press, San Diego, pp. 373–402.

    Google Scholar 

  49. T. French (1996). The Development of Fluorescence Lifetime Imaging and an Application in Immunology, PhD Thesis. University of Illinois at Urbana–Champaign.

  50. W. Becker and A. Bergmann (2002). Detectors for High-Speed Photon Counting, The TCSPC Knowledge Base, Berlin. Also availabe at http://www.becker-hickl.de/pdf/spcdetect1.pdf.

  51. K. Berndt (1985). Application of gain-modulated avalanche photodiodes in phase-sensitive fluorescence spectroscopy. Optics Commun. 56, 30–35.

    Google Scholar 

  52. J. Pouget, J. Mugnier, and B. Valeur (1989). Correction of timing errors in multifrequency phase(modulation fluorometry., J. Phys. E: Sci. Instrum. 22, 855–862.

    Google Scholar 

  53. P. Gauduchon and Ph. Wahl (1978). Pulse fluorimetry of tyrosyl peptides. Biophys. Chem. 8, 87–104; R. W. Wijnaendts van Resandt, R. H. Vogel, and S. W. Provencher (1982). Double beam fluorescence lifetime spectrometer with subpicosecond resolution: Application to aqueous tryptophan. Rev. Sci. Instrum. 53, 1392–1397; M. Zuker, A. G. Szabo, L. Bramall, D. T. Krajcarski, and B. Selinger (1985). Delta function convolution method (DFCM) for fluorescence decay experiments. Rev. Sci. Instrum. 56, 14–22; M. Van den Zegel, N. Boens, D. Daems, and F. C. De Schryver (1986). Possibilities and limitations of the time-correlated single photon counting technique: A comparative study of correction methods for the wavelength dependence of the instrument response function. Chem. Phys. 101, 311–335; N. Boens, M. Ameloot, I. Yamazaki, and F. C. De Schryver (1988). On the use and the performance of the delta function convolution method for the estimation of fluorescence decay parameters. Chem. Phys. 121, 73–86.

  54. N. Boens, N. Tamai, I. Yamazaki, and T. Yamazaki (1990). Picosecond single photon timing measurements with a proximity type microchannel plate photomultiplier and global analysis with reference convolution. Photochem. Photobiol. 52, 911– 917.

    Google Scholar 

  55. R. E. Imhof and D. J. S. Birch (1982). Distortion of gaussian pulses by a diffraction grating. Optics Commun. 42, 83–86.

    Google Scholar 

  56. R. V. Krishnan, H. Saitoh, H. Terada, V. E. Centonze, and B. Herman (2003). Development of a multiphoton fluorescence lifetime imaging microscope system using a streak camera. Rev. Sci. Instrum. 74, 2714–2721.

    Google Scholar 

  57. K. Kemnitz, L. Pfeifer, and M. R. Ainbund (1997). Detector for multichannel spectroscopy and fluorescence lifetime imaging on the picosecond timescale. Nucl. Instrum. Methods Phys. Res, Sect. A, 387, 86–87.

    Google Scholar 

  58. F. R. Boddeke (1998). Quantitative Fluorescence Microscopy, Dissertation, Delft Technical University.

  59. J. M. Beechem, E. James, and L. Brand (1990). Time-resolved fluorescence studies of the protein folding process: New instrumentation, analysis and experimental approaches. In J. R. Lakowicz (Ed.), Time-Resolved Laser Spectroscopy in Biochemistry II. SPIE Proceedings, Vol. 1204, Los Angeles, pp. 686– 698.

  60. J. M. Beechem (1992). Global analysis of biophysical data. In L. Brand and M. L. Johnson (Eds.), Methods in Enzymology, Vol. 210, Academic Press, San Diego, pp. 37–54.

    Google Scholar 

  61. P. J. Verveer and P. I. H. Bastiaens (2003). Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data. J. Microsc. Oxford, 209, 1–7.

    Google Scholar 

  62. J. M. Beechem, M. Ameloot, and L. Brand (1985). Global analysis of fluorescence decay surfaces—Excited state reactions. Chem. Phys. Lett. 120, 466–472; M. Ameloot, J. M. Beechem, and L. Brand (1986). Compartmental modeling of excited state reactions. Identifiability of the rate constants from fluorescence decay surfaces. Chem. Phys. Lett. 129, 211–219; M. Ameloot, N. Boens, R. Andriessen, V. Van den Bergh, and F. C. De Schryver (1991). Non a priori analysis of the fluorescence decay surfaces of excited-state processes. 1: Theory. J. Phys. Chem. 95, 2041–2047; M. Ameloot, N. Boens, R. Andriessen, V. Van den Bergh, and F. C. De Schryver (1992). Compartmental analysis of fluorescence decay surfaces of excited-state processes. In L. Brand and M. L. Johnson (Eds.), Methods in Enzymology, Vol. 210, Academic Press, San Diego, pp. 314– 340.

  63. D. F. Eaton (1990). Recommended methods for fluorescence decay analysis. Pure Appl. Chem. 62, 1631–1648.

    Google Scholar 

  64. D. Marquardt (1963). An algorithm for least squares estimation of nonlinear parameters, J. Appl. Math. 11, 431–441.

    Google Scholar 

  65. N. Boens, M. Van den Zegel, and F. C. De Schryver (1984). Picosecond determination of the second excited singlet lifetime of xanthione in solution. Chem. Phys. Lett. 111, 340–346; N. Boens, M. Van den Zegel, and F. C. De Schryver (1984). ibid. 113, 602.

  66. D. R. James and W. R. Ware (1985). A fallacy in the interpretation of fluorescence decay parameters. Chem. Phys. Lett. 120, 455– 459.

    Google Scholar 

  67. J. C. Brochon (1994). Maximum entropy method of data analysis in time-resolved spectroscopy. In L. Brand and M. L. Johnson (Eds.), Methods in Enzymology, Vol. 240, Academic Press, San Diego, pp. 262–311.

  68. Siemiarczuk, B. D. Wagner, and W. R. Ware (1990). Comparison of the maximum entropy and exponential series methods for the recovery of distributions of lifetimes from fluorescence lifetime data. J. Phys. Chem. 94, 1661–1666.

  69. Y. S. Liu and W. R. Ware (1993). Photophysics of polycyclic aromatic hydrocarbons adsorbed on silica gel surfaces. 1: Fluorescence lifetime distribution analysis. An ill-conditioned problem. J. Phys. Chem. 97, 5980– 5986.

    Google Scholar 

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Authors and Affiliations

  1. Biomedisch Onderzoeksinstituut, Limburgs Universitair Centrum, School of Life Sciences, Transnationale Universiteit Limburg, 3590, Diepenbeek, Belgium

    Martin vandeVen & Marcel Ameloot

  2. CNRS UMR 8531, Laboratoire de Chimie générale, CNAM, 292 rue Saint-Martin, 75141, Paris cedex 03, France

    Bernard Valeur

  3. CNRS UMR 8531, Laboratoire PPSM, ENS-Cachan, 61 avenue du Président Wilson, 94235, Cachan cedex, France

    Bernard Valeur

  4. Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200 F, 3001, Heverlee, Belgium

    Noël Boens

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  1. Martin vandeVen
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  4. Noël Boens
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vandeVen, M., Ameloot, M., Valeur, B. et al. Pitfalls and Their Remedies in Time-Resolved Fluorescence Spectroscopy and Microscopy. J Fluoresc 15, 377–413 (2005). https://doi.org/10.1007/s10895-005-2632-1

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