Pharmaceutical Research

, Volume 16, Issue 8, pp 1153–1162 | Cite as

Fluorescence Recovery After Photobleaching: A Versatile Tool for Mobility and Interaction Measurements in Pharmaceutical Research

  • Tom K. L. Meyvis
  • Stefaan C. De SmedtEmail author
  • Patrick Van Oostveldt
  • Joseph Demeester


This review introduces the basics of fluorescence recovery after photobleaching (FRAP) from a theoretical and an instrumentational approach. The most interesting and innovative applications with a pharmaceutical point of view are briefly discussed and possible future applications are suggested. These future applications include research on the mobility of macromolecular drugs in macro- or microscopic pharmaceutical dosage forms, mobility, and binding of antitumor drugs in tumor tissue, intracellular trafficking of gene complexes and mobility of drugs in membranes prior to transmembrane penetration. The paper is also intended to be an introductory guideline to those who would like to get involved in FRAP related experimental techniques. Therefore, comprehensive details on different setups and data analysis are given, as well as a brief outline of the problems that may be encountered when performing FRAP. Overall, this review shows the great potential of FRAP in pharmaceutical research. This is complemented by our own results illustrating the possibility of performing FRAP in microscopic dosage forms (microspheres) using a high resolution variant of FRAP.

FRAP mobility interactions diffusion drug delivery 


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  1. 1.
    J. C. G. Blonk, A. Don, H. Van Aalst, and J. J. Birmingham. Fluorescence photobleaching recovery in the confocal scanning light microscope. J. Microsc. 169:363-374 (1993).Google Scholar
  2. 2.
    U. Kubitscheck, M. Tschödrich-Rotter, P. Wedekind, and R. Peters. Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements. J. Microsc. 182:225-233 (1996).Google Scholar
  3. 3.
    Axelrod, D. E. Koppel, J. Chlessinger, J. Elson, and W. W. Webb. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16:1055-1069 (1976).Google Scholar
  4. 4.
    R. Swaminathan, S. Bicknese, N. Periasamy, and A. S. Verkman. Cytoplasmatic viscosity near the cell plasma membrane: Translational diffusion of small fluorescent solute measured by Total Internal Reflection-Fluorescence Photobleaching Recovery. Biophys. J. 71:1140-1151 (1996).Google Scholar
  5. 5.
    M. Velez and D. Axelrod. Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes. Biophys. J. 53:575-591 (1988).Google Scholar
  6. 6.
    P. Wedekind, U. Kubitscheck, and R. Peters. Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging. J. Microsc. 176:23-33 (1994).Google Scholar
  7. 7.
    D. M. Soumpasis. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys. J. 41:95-97 (1983).Google Scholar
  8. 8.
    G. W. Gordon, B. Chazotte, X. F. Wang, and B. Herman. Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components. Biophys. J. 68:766-778 (1995).Google Scholar
  9. 9.
    A. Lopez, L. Dupou, A. Altibelli, J. Trotard, and J. F. Tocanne. Fluorescence recovery after photobleaching (FRAP) experiments under conditions of uniform disk illumination. Critical comparison of analytical solutions, and a new mathematical method for calculation of diffusion coefficient D. Biophys. J. 53:963-970 (1988).Google Scholar
  10. 10.
    R. Peters. Translational diffusion in plasma membrane of single cells as studied by fluorescence microphotolysis. Cell. Biol. Int. Rep. 5:733-760 (1981).Google Scholar
  11. 11.
    P. Wedekind, U. Kubitscheck, O. Heinrich, and R. Peters. Line-Scanning Microphotolysis for Diffraction-Limited Measurements of Lateral Diffusion. Biophys. J. 71:1621-1632 (1996).Google Scholar
  12. 12.
    T. Tsay and K. A. Jacobson. Spatial Fourier analysis of video photobleaching measurements, principles and optimization. Biophys. J. 60:360-368 (1991).Google Scholar
  13. 13.
    D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain. Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media. Biophys. J. 65:2428-2436 (1993).Google Scholar
  14. 14.
    B. A. Westrin, A. Axelsson, and G. Zacchi. Diffusion measurement in gels. J. Contr. Rel. 30:189-199 (1994).Google Scholar
  15. 15.
    W. M. Saltzman, M. L. Radomsky, K. J. Whaley, and R. A. Cone. Antibody diffusion in human cervical mucus. Biophys. J. 66:508-515 (1994).Google Scholar
  16. 16.
    J. D. Bryers and F. Drummond. Local mass transfer coefficients in bacterial biofilms using fluorescence recovery after photobleaching (FRAP). In R. H. Wijffels, R. M. Buitelaar, C. Bucke, and J. Tramper (eds), Immobilized cells: Basics and applications, Elsevier, Amsterdam, 1996, pp. 196-204.Google Scholar
  17. 17.
    M. Moussaoui, M. Benlyas, and P. Wahl. Diffusion of proteins in Sepharose CL-B gels. J. Chromatogr. 591:115-120 (1995).Google Scholar
  18. 18.
    E. M. Johnson, D. A. Berk, R. K. Jain, and W. M. Deen. Diffusion and partitioning of proteins in charged agarose gels. Biophys. J. 68:1561-1568 (1995).Google Scholar
  19. 19.
    Z. Bu and P. S. Russo. Diffusion of dextran in aqueous (hydroxypropyl) cellulose. Macromolecules 27:1187-1194 (1994).Google Scholar
  20. 20.
    B. Tinland and R. Borsali. Single-chain diffusion coefficient of F-dextran in poly (vinylpyrrolidone) water: fluorescence recovery after photobleaching experiments. Macromolecules 27:2142-2144 (1994).Google Scholar
  21. 21.
    S. Pajevic, R. Bansil, and C. Konak. Diffusion of linear polymer chains in methyl methacrylate gels. Macromolecules 26:305-312 (1993).Google Scholar
  22. 22.
    S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. Van Oostveldt, J. C. G. Blonk, and W. E. Hennink. Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy. Macromolecules 30:4863-4870 (1997).Google Scholar
  23. 23.
    O. Seksek, J. Biwersi, and A S. Verkman. Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell Biol. 138:131-142 (1997).Google Scholar
  24. 24.
    R. Swaminathan, C. P. Hoang, and A. S. Verkman. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys. J. 72:1900-1907 (1997).Google Scholar
  25. 25.
    S. R. Chary and R. K. Jain. Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. Proc. Natl. Acad. Sci. USA 86:5385-5389 (1989).Google Scholar
  26. 26.
    G. Molema, L. F. M. H. de Leij, and D. K. F. Meijer. Tumor vascular endothelium: barrier or target in tumor directed drug delivery and immunotherapy. Pharm. Res. 14:2-10 (1997).Google Scholar
  27. 27.
    Y. I. Henis. Lateral and rotational diffusion in biological membranes. In M. Shinitzky (ed), Biomembranes: Physical Aspects, VCH Publishers, Weinheim-New York, 1998, pp. 279-340.Google Scholar
  28. 28.
    T. J. Feder, I. Brust Mascher, J. P. Slattery, B. Baird, and W. W. Webb. Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys. J. 70:2767-2773 (1996).Google Scholar
  29. 29.
    M. E. Johnson, D. A. Berk, D. Blankschtein, D. E. Golan, R. K. Jain, and R. S. Langer. Lateral diffusion of small compounds in human stratum corneum and model lipid bilayer systems. Biophys. J. 71:2656-2668 (1996).Google Scholar
  30. 30.
    D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain. Direct in vivo measurement of targeted binding in a human tumor xenograft. Proc. Natl. Acad. Sci. USA 94:1785-1790 (1997).Google Scholar
  31. 31.
    E. N. Kaufman and R. K. Jain. Measurement of mass transport and reaction parameters in bulk solution using photobleaching. Reaction limited binding regime. Biophys. J. 60:596-610 (1991).Google Scholar
  32. 32.
    E. N. Kaufman and R. K. Jain. In vitro measurement and screening of monoclonal antibody affinity using fluorescence photobleaching. J. Immunol. Meth. 155:1-17 (1992).Google Scholar
  33. 33.
    B. Flamion, P. M. Bungay, C. C. Gibson, and K. R. Spring. Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery. Biophys. J. 60:1229-1242 (1991).Google Scholar
  34. 34.
    D. A. Berk, M. A. Swartz, A. J. Leu, and R. K. Jain. Transport in lymphatic capillaries. II. Microscopic velocity measurement with fluorescence photobleaching. Am. J. Physiol. 270:330-337 (1996).Google Scholar
  35. 35.
    M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington. Crystal structure of Aequorea victoria green fluorescent protein. Science 273:1392-1395 (1996).Google Scholar
  36. 36.
    C. Dietrich, R. Merkel, and R. Tampé. Diffusion measurement of fluorescence labeled amphiphilic molecules with a standard fluorescence microscope. Biophys. J. 72:1701-1710 (1997).Google Scholar
  37. 37.
    J. L. Robeson and R. D. Tilton. Effect of concentration quenching on fluorescence recovery after photobleaching measurements. Biophys. J. 68:2145-2155 (1995).Google Scholar
  38. 38.
    N. Periasamy, S. Bicknese, and A. S. Verkman. Reversible photobleaching of fluorescein conjugates in air-saturated viscous solutions: Singlet and triplet state quenching by tryptophan. Photochem. and Photobiol. 63:265-271 (1996).Google Scholar
  39. 39.
    S. C. De Smedt, A. Lauwers, J. Demeester, Y. Engelborghs, G. De Mey, and M. Du. Structural information on hyaluronic acid solutions as studied by probe diffusion experiments. Macromolecules 27:141-146 (1994).Google Scholar

Copyright information

© Plenum Publishing Corporation 1999

Authors and Affiliations

  • Tom K. L. Meyvis
    • 1
  • Stefaan C. De Smedt
    • 1
    Email author
  • Patrick Van Oostveldt
    • 2
  • Joseph Demeester
    • 1
  1. 1.Laboratory of General Biochemistry and Physical PharmacyUniversity of GentGentBelgium
  2. 2.Laboratory of Biochemistry and Molecular CytologyUniversity of GentGentBelgium

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