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Instrumentation for Fluorescence Spectroscopy

  • Joseph R. Lakowicz

Abstract

The successful application of fluorescence methods requires considerable attention to experimental details and a good understanding of the instrumentation. There are numerous potential artifacts which can distort the data. Fluorescence is a highly sensitive method. The gain or amplification of the instruments can usually be increased to obtain observable signals, even if the sample is nonfluorescent. These signals seen at high amplification may not originate with the fluorophore of interest. Instead, one may observe interference due to background fluorescence from the solvents, light leaks in the instrumentation, stray light passing through the optics, light scattered by turbid solutions, Rayleigh scatter, and/or Raman scatter, to name a few sources of interference. Furthermore, there is no ideal spectrofluorometer, and the available instruments do not yield true excitation or emission spectra. This is because of the nonuniform spectral output of the light sources and the wavelength-dependent efficiency of the monochromators and detectors (photomultiplier tubes). The polarization or anisotropy of the emitted light can also affect the measured fluorescence intensities. To obtain reliable spectral data, one needs to be aware of and control these numerous factors. In this chapter we will discuss the properties of the individual components in a spectrofluorometer and how these properties affect the observed spectral data.

Keywords

Emission Spectrum Fluorescence Spectroscopy Xenon Lamp Stray Light Exciting Light 
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.

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References

  1. 1.
    Technical Literature, Oriel Instruments, 250 Long Beach Blvd., PO Box 872, Stratford, CT 06497.Google Scholar
  2. 2.
    Laczko, G., and Lakowicz, J. R., unpublished observations.Google Scholar
  3. 3.
    Light Sources, Monochromators & Spectrographs, Detectors & Detection Systems, Fiber Optics, Oriel Corporation, 250 Long Beach Blvd., PO Box 872, Stratford, CT 06497.Google Scholar
  4. 4.
    Cermax Product Specifications for Collimated and Focused Xenon Lamps, ILC Technology, Inc., 399 West Joan Drive, Sunnyvale, CA 94089.Google Scholar
  5. 5.
    Analamp Emission Data Sheet, BHK, Inc. (Subsidiary of Hamamatsu Corporation), 1000 S. Magnolia Ave., Monrovia, CA 91016.Google Scholar
  6. 6.
    Sipior, J., Carter, G. M., Lakowicz, J. R., and Rao, G., 1997, Blue light-emitting diode demonstrated as an ultraviolet excitation source for nanosecond phase-modulation fluorescence lifetime measurements, Rev. Sci. Instrum. 68: 2666 – 2670.CrossRefGoogle Scholar
  7. 7.
    1996 Catalog of Optical Components and Instruments, Optometrics USA, Inc., Nemco Way, Stony Brook Industrial Park, Ayer, MA 01432.Google Scholar
  8. 8.
    Gryczynski, I., and Lakowicz, J. R., unpublished observations.Google Scholar
  9. 9.
    Castellano, P., and Lakowicz, J. R., unpublished observations.Google Scholar
  10. 10.
    Technical Literature, Spindler & Hoyer, Inc., 459 Fortune Blvd., Milford, MA 01757.Google Scholar
  11. 11.
    Flaugh, P. L., O'Donnell, S. E., and Asher, S. A., 1984, Development of a new optical wavelength rejection filter: Demonstration of its utility in Raman spectroscopy, Appl. Spectrosc. 386: 847 – 850.CrossRefGoogle Scholar
  12. 12.
    Gryczynski, I., Malak, H., Lakowicz, J. R., Cheung, H. C., Robinson, J., and Umeda, P. K., 1996, Fluorescence spectral properties of troponin C mutant F22W with one-, two-and three-photon excitation, Biophys. J. 71: 3448 – 3453.CrossRefGoogle Scholar
  13. 13.
    Szmacinski, H., Gryczynski, I., and Lakowicz, J. R., 1993, Calcium-dependent fluorescence lifetimes of Indo-1 for one-and two-photon excitation of fluorescence, Photochem. Photobiol. 58: 341 – 345.CrossRefGoogle Scholar
  14. 14.
    Photomultiplier Tubes, Hamamatsu Photonics K.K., Electron Tube Center, (1994), 314-5, Shimokanzo, Toyooka-village, Iwata-gun, Shizuoka-ken, 438–01 Japan.Google Scholar
  15. 15.
    Leaback, D. H., 1997, Extended theory, and improved practice for the quantitative measurement of fluorescence, J. Fluoresc. 7 (1): 55S – 57S.CrossRefGoogle Scholar
  16. 16.
    Hiraoka, Y., Sedat, J. W., and Agard, D. A., 1987, The use of a charge-coupled device for quantitative optical microscopy of biological structures, Science 238: 36 – 41.CrossRefGoogle Scholar
  17. 17.
    Aikens, R. S., Agard, D. A., and Sedat, J. W., 1989, Solid-state imagers for microscopy, Methods Cell Biol. 29: 291 – 313.CrossRefGoogle Scholar
  18. 18.
    Epperson, P. M., and Denton, M. B., 1989, Binding spectral images in a charge-coupled device, Anal. Chem. 61: 1513 – 1519.CrossRefGoogle Scholar
  19. 19.
    Bilhorn, R. B., Sweedler, J. V., Epperson, R M., and Denton, M. B., 1987, Charge transfer device detectors for analytical optical spectroscopy-operation and characteristics, Appl. Spectrosc. 41: 1114 – 1124.CrossRefGoogle Scholar
  20. 20.
    Epperson, P. M., Jalkaian, R. D., and Denton, M. B., 1989, Molecular fluorescence measurements with a charge-coupled device detector, Anal. Chem. 61: 282 – 285.CrossRefGoogle Scholar
  21. 21.
    Melhuish, W. H., 1962, Calibration of spectrofluorometers for measuring corrected emission spectra, J. Opt. Soc. Am. 52: 1256 – 1258.CrossRefGoogle Scholar
  22. 22.
    Yguerabide, J., 1968, Fast and accurate method for measuring photon flux in the range 2500-6000 A, Rev. Sci. Instrum. 397: 1048 – 1052.Google Scholar
  23. 23.
    Mandal, K., Pearson, T. D. L., and Demas, J. N., 1980, Luminescent quantum counters based on organic dyes in polymer matrices, Anal. Chem. 52: 2184 – 2189.CrossRefGoogle Scholar
  24. 24.
    Mandat, K., Pearson, T. D. L., and Demas, N. J., 1981, New luminescent quantum counter systems based on a transition-metal complex, Inorg. Chem. 20: 786 – 789.CrossRefGoogle Scholar
  25. 25.
    Nothnagel, E. A., 1987, Quantum counter for correcting fluorescence excitation spectra at 320- and 800-nm wavelengths, Anal. Biochem. 163: 224 – 237.CrossRefGoogle Scholar
  26. 26.
    Lippert, E., Nagelle, W., Siebold-Blakenstein, I., Staiger, U., and Voss, W., 1959, Messung von fluorescenzspektren mit hilfe von spektralphotometern und vergleichsstandards, Z Anal. Chem. 17:1– 18.Google Scholar
  27. 27.
    Schmillen, A., and Legler, R., 1967, Landoll-Bornstein, Vol. 3, Lumineszenz Organischer Substanzen, Springer-Verlag, New York, pp. 228 – 229.Google Scholar
  28. 28.
    Argauer, R. J., and White, C. E., 1964, Fluorescent compounds for calibration of excitation and emission units of spectrofluorometer, Anal. Chem. 36: 368 – 371.CrossRefGoogle Scholar
  29. 29.
    Melhuish, W. H., 1960, A standard fluorescence spectrum for calibrating spectrofluorometers, J. Phys. Chem. 64: 762 – 764.CrossRefGoogle Scholar
  30. 30.
    Parker, C. A., 1962, Spectrofluorometer calibration in the ultraviolet region, Anal. Chem. 34: 502 – 505.CrossRefGoogle Scholar
  31. 31.
    Velapoldi, R. A., 1973, Considerations on organic compounds in solution and inorganic ions in glasses as fluorescent standard reference materials, Proc. Natl. Bur. Stand. 378: 231 – 244.Google Scholar
  32. 32.
    Pardo, A., Reyman, D., Poyato, J. M. L., and Medina, E, 1992, Some (ì-carboline derivatives as fluorescence standards, J. Lumin. 51: 269274.Google Scholar
  33. 33.
    Chen, R. F., 1967, Some characteristics of the fluorescence of quinine, Anal. Biochem. 19: 374 – 387.CrossRefGoogle Scholar
  34. 34.
    Verity, B., and Bigger, S. W., 1996, The dependence of quinine fluorescence quenching on ionic strength, Int. J. Chem. Kinet. 2812: 919 – 923.CrossRefGoogle Scholar
  35. 35.
    Ghiggino, K. P., Ski1ton, P. F., and Thistlethwaite, P. J., 1985, (i-Carboline as a fluorescence standard, J. Photochem. 31: 113 – 121.Google Scholar
  36. 36.
    Middleton, W. E. K., and Sanders, C. L., 1951, The absolute spectral diffuse reflectance of magnesium oxide, J. Opt. Soc. Am. 41(6): 419–424.Google Scholar
  37. 37.
    Heller, C. A., Henry, R. A., McLaughlin, B. A., and Bliss, D. E., 1974, Fluorescence spectra and quantum yields: Quinine, uranine, 9,10-diphenylanthracene, and 9,10–bis(phenylethynyl)anthracenes, J. Chem. Eng. Data 19 (3): 214 – 219.CrossRefGoogle Scholar
  38. 38.
    Melhuish, W. H., 1972, Absolute spectrofluorometry, J. Res. of the National Bureau of Standards 76A: 547 – 560.CrossRefGoogle Scholar
  39. 39.
    Tazuke, S., and Winnik, M. A., 1986, Fluorescence and phosphorescence spectroscopy in polymer systems: A general introduction, in Photophysical and Photochemical Tools in Polymer Science, M. A. Winnik (ed.), D. Reidel, Dordrecht, pp. 15 – 42.CrossRefGoogle Scholar
  40. 40.
    Demas, J. N., and Crosby, G. A., 1971, The measurement of photoluminescence quantum yields. A review, J. Phys. Chem. 75: 991 1025.Google Scholar
  41. 41.
    Birks, J. B., 1970, Photophysics of Aromatic Molecules, Wiley-Interscience, New York, p. 98.Google Scholar
  42. 42.
    Hermans, J. J., and Levinson, S., 1951, Some geometrical factors in light-scattering apparatus, J. Opt. Soc. Am. 41 (7): 460 – 465.CrossRefGoogle Scholar
  43. 43.
    Eastman, J. W., 1967, Quantitative spectrofluorimetry—the fluorescence quantum yield of quinine sulfate, Photochem. Photobiol. 6: 55 – 72.CrossRefGoogle Scholar
  44. 44.
    Adams, M. J., Highfield, J. G., and Kirkbright, G. E, 1977, Determination of absolute fluorescence quantum efficiency of quinine bisulfate in aqueous medium by optoacoustic spectrometry, Anal. Chem. 49: 1850 – 1852.CrossRefGoogle Scholar
  45. 45.
    Brannon, J. H., and Magde, D., 1978, Absolute quantum yield determination by thermal blooming. Fluorescein, J. Phys. Chem. 82: 705 – 709.CrossRefGoogle Scholar
  46. 46.
    Mardelli, M., and Olmsted, J., 1977, Calorimetric determination of the 9,10-diphenyl-anthracene fluorescence quantum yield, J. Photochem. 7: 277 – 285.CrossRefGoogle Scholar
  47. 47.
    Ware, W. R., and Rothman, W., 1976, Relative fluorescence quantum yields using an integrating sphere. The quantum yield of 9,10diphenylanthracene in cyclohexane, Chem. Phys. Lett. 39: 449 – 453.CrossRefGoogle Scholar
  48. 48.
    Testa, A. C., 1969, Fluorescence quantum yields and standards, Fluorescence News; Newsletter on Luminescence 4 (4): 1 – 3.Google Scholar
  49. 49.
    Rusakowicz, R., and Testa, A. C., 1968, 2–Aminopyridine as a standard for low-wavelength spectrofluorometry, J. Phys. Chem. 72: 2680 – 2681.Google Scholar
  50. 50.
    Chen, R. E, 1967, Fluorescence quantum yields of tryptophan and tyrosine, Anal. Lett. 1: 35 – 42.CrossRefGoogle Scholar
  51. 51.
    Fischer, M., and Georges, J., 1996, Fluorescence quantum yield of rhodamine 6G in ethanol as a function of concentration using thermal lens spectrometry, Chem. Phys. Lett. 260: 115 – 118.CrossRefGoogle Scholar
  52. 52.
    Karstens, T., and Kobe, K., 1980, Rhodamine B and Rhodamine 101 as reference substances for fluorescence quantum yield measurements, J. Phys. Chem. 84: 1871 – 1872.CrossRefGoogle Scholar
  53. 53.
    Magde, D., Brannon, J. H., Cremers, T. L., and Olmsted, J., 1979, Absolute luminescence yield of cresyl violet. A standard for the red, J. Phys. Chem. 83: 696 – 699.CrossRefGoogle Scholar
  54. 54.
    Kubista, M., Sjöback, R., Eriksson, S., and Albinsson, B., 1994, Experimental correction for the inner-filter effect in fluorescence spectra, Analyst 119: 417 – 419.CrossRefGoogle Scholar
  55. 55.
    Yappert, M. C., and Ingle, J. D., 1989, Correction of polychromatic luminescence signals for inner-filter effects, Appl. Spectrosc. 43: 759 – 767.CrossRefGoogle Scholar
  56. 56.
    Wiechelman, K. J., 1986, Empirical correction equation for the fluorescence inner filter effect, Am. Lab. 18: 49 – 53.Google Scholar
  57. 57.
    Puchalski, M. M., Mora, M. J., and von Wandruszka, R., 1991, Assessment of inner filter effect corrections in fluorimetry, Fresenius J. Anal. Chem. 340: 341 – 344.CrossRefGoogle Scholar
  58. 58.
    Guilbault, G. G. (ed.), 1990, Practical Fluorescence, Marcel Dekker, New York, p. 31.Google Scholar
  59. 59.
    Eisinger, J., 1969, A variable temperature, U.V. luminescence spectrograph for small samples, Photochem. Photobiol. 9: 247 – 258.CrossRefGoogle Scholar
  60. 60.
    Eisinger, J., and Flores, J., 1979, Front-face fluorometry of liquid samples, Anal. Biochem. 94: 15 – 21.CrossRefGoogle Scholar
  61. 61.
    Berlman, I. B., 1971, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York.Google Scholar
  62. 62.
    Kasha, M., 1960, Paths of molecular excitation, Radiat. Res. 2: 243–275.Google Scholar
  63. 63.
    Gryczynski, I., unpublished observations.Google Scholar
  64. 64.
    Lakowicz, J. R., Gryczynski, I., Kulba, J., and Danielsen, E., 1992, Two photon induced fluorescence intensity and anisotropy decays of diphenylhexatriene in solvents and lipid bilayers, J. Fluoresc. 2 (4): 247 – 258.CrossRefGoogle Scholar
  65. 65.
    Xu, C., and Webb, W. W., 1997, Multiphoton excitation of molecular fluorophore and nonlinear laser microscopy, in Topics in Fluorescence Spectroscopy, Volume 5, Nonlinear and Two-Photon-Induced Fluorescence, J. R. Lakowicz (ed.), Plenum Press, New York, pp. 471 – 540.Google Scholar
  66. 66.
    Callis, P. R., 1997, Two-photon induced fluorescence, Annu. Rev. Phys. Chem. 48: 271 – 297.CrossRefGoogle Scholar
  67. 67.
    Bowman, R. L., Caulfield, P. A., and Udenfriend, S., 1955, Spectrophotofluorometric assay in the visible and ultraviolet, Science 122: 32 – 33.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

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