OSA's Digital Library

Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editor: Gregory W. Faris
  • Vol. 1, Iss. 12 — Dec. 18, 2006

Confocal fluorescence polarization microscopy in turbid media: effects of scattering-induced depolarization

Chad E. Bigelow and Thomas H. Foster  »View Author Affiliations

JOSA A, Vol. 23, Issue 11, pp. 2932-2943 (2006)

View Full Text Article

Enhanced HTML    Acrobat PDF (315 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We present an experimental and theoretical study of confocal fluorescence polarization microscopy in turbid media. We have performed an experimental study using a fluorophore-embedded polymer rod immersed in aqueous suspensions of 0.1 and 0.5 μ m diameter polystyrene microspheres. A Monte Carlo approach to simulate confocal fluorescence polarization imaging in scattering media is also presented. It incorporates a detailed model of polarized fluorescence generation that includes sampling of elliptical polarization, excited-state molecular rotational Brownian motion, and dipole fluorescence emission. Using both approaches, we determine the effects of the number of scattering events, target depth, photon scattering statistics, objective numerical aperture, and pinhole size on confocal anisotropy imaging. From this detailed analysis and comparison of experiment with simulation, we determine that fluorescence polarization is maintained to depths at which meaningful intensity images can be acquired.

© 2006 Optical Society of America

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy
(260.5430) Physical optics : Polarization
(290.4210) Scattering : Multiple scattering
(290.7050) Scattering : Turbid media

ToC Category:

Original Manuscript: January 3, 2006
Revised Manuscript: May 4, 2006
Manuscript Accepted: May 16, 2006

Virtual Issues
Vol. 1, Iss. 12 Virtual Journal for Biomedical Optics

Chad E. Bigelow and Thomas H. Foster, "Confocal fluorescence polarization microscopy in turbid media: effects of scattering-induced depolarization," J. Opt. Soc. Am. A 23, 2932-2943 (2006)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum, 1999).
  2. A. W. Knight, N. J. Goddard, N. Billinton, P. A. Cahill, and R. M. Walmsley, "Fluorescence polarization discriminates green fluorescent protein from interfering autofluorescence in a microplate assay for genotoxicity," J. Biochem. Biophys. Methods 51, 165-177 (2002). [CrossRef] [PubMed]
  3. A. W. Knight, N. J. Goddard, P. R. Fielden, M. G. Barker, N. Billinton, and R. M. Walmsley, "Fluorescence polarisation of green fluorescent protein (GFP). A strategy for improved wavelength discrimination for GFP determinations," Anal. Commun. 36, 113-117 (1999). [CrossRef]
  4. S. M. Blackman, C. E. Cobb, A. H. Beth, and D. W. Piston, "The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy," Biophys. J. 71, 194-208 (1996). [CrossRef] [PubMed]
  5. T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005). [CrossRef] [PubMed]
  6. C. E. Bigelow, H. D. Vishwasrao, J. G. Frelinger, and T. H. Foster, "Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy," J. Microsc. 215, 24-33 (2004). [CrossRef] [PubMed]
  7. M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, "Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells," Biophys. J. 78, 2614-2627 (2000). [CrossRef] [PubMed]
  8. A. Diaspro, G. Chirico, F. Federici, F. Cannone, S. Beretta, and M. Robello, "Two-photon microscopy and spectroscopy based on a compact confocal scanning head," J. Biomed. Opt. 6, 300-310 (2001). [CrossRef] [PubMed]
  9. I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001). [CrossRef] [PubMed]
  10. Y. Wu, F. F. Sun, D. M. Tong, and B. M. Taylor, "Changes in membrane properties during energy depletion-induced cell injury studied with fluorescence microscopy," Biophys. J. 71, 91-100 (1996). [CrossRef] [PubMed]
  11. I. I. Smalyukh, S. V. Shiyanovskii, and O. D. Lavrentovich, "Three-dimensional imaging of orientational order by fluorescence confocal polarizing microscopy," Chem. Phys. Lett. 336, 88-96 (2001). [CrossRef]
  12. A. Entwistle and M. Noble, "The use of polarization analysis in the quantification of fluorescent emission: general principles," J. Microsc. 165, 331-346 (1992). [CrossRef]
  13. T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).
  14. X. Gan and M. Gu, "Effective point-spread function for fast image modeling and processing in microscopic imaging through turbid media," Opt. Lett. 24, 741-743 (1999). [CrossRef]
  15. S. P. Schilders and M. Gu, "Limiting factors on image quality in imaging through turbid media under single-photon and two-photon excitation," Microsc. Microanal. 6, 156-160 (2000). [PubMed]
  16. B. F. Dickens, T. R. Snow, V. Green, and W. B. Weglicki, "The effect of erythrocyte associated light scattering on membrane fluorescence polarization," Mol. Cell. Biochem. 79, 91-94 (1988). [CrossRef] [PubMed]
  17. F. W. J. Teale, "Fluorescence depolarization by light-scattering in turbid solutions," Photochem. Photobiol. 10, 363-374 (1969). [CrossRef] [PubMed]
  18. J. M. Schmitt, A. Knüttel, and M. Yadlowsky, "Confocal microscopy in turbid media," J. Opt. Soc. Am. A 11, 2226-2235 (1994). [CrossRef]
  19. C. E. Bigelow, "Biological applications of confocal fluorescence polarization microscopy" (University of Rochester, Rochester, N.Y., 2005), http://hdl.handle.net/1802/2358.
  20. C. E. Bigelow, D. L. Conover, and T. H. Foster, "Confocal fluorescence spectroscopy and anisotropy imaging system," Opt. Lett. 28, 695-697 (2003). [CrossRef] [PubMed]
  21. C. E. Bigelow, C. J. Harkrider, D. L. Conover, T. H. Foster, I. Georgakoudi, S. Mitra, M. G. Nichols, and M. Rajadhyaksha, "Retrofitted confocal laser scanner for a commercial inverted fluorescence microscope," Rev. Sci. Instrum. 72, 3407-3410 (2001). [CrossRef]
  22. J. D. Wilson, C. E. Bigelow, D. J. Calkins, and T. H. Foster, "Light scattering from intact cells reports oxidative-stress-induced mitochondrial swelling," Biophys. J. 88, 2929-2938 (2005). [CrossRef] [PubMed]
  23. T. Wilson, Confocal Microscopy (Academic, 1990).
  24. S. C. Harvey and H. C. Cheung, "Computer simulation of fluorescence depolarization due to Brownian motion," Proc. Natl. Acad. Sci. U.S.A. 69, 3670-3672 (1972). [CrossRef] [PubMed]
  25. L. V. Wang, S. L. Jacques, and L. Zheng, "MCML: Monte Carlo modeling of photon transport in multi-layered tissues," Comput. Methods Programs Biomed. 47, 131-146 (1995). [CrossRef] [PubMed]
  26. S. A. Prahl, M. Keijzer, S. L. Jacques, and A. J. Welch, A Monte Carlo Model of Light Propagation in Tissue, Proc. SPIE Institute Series Vol. IS 5 (SPIE, 1989), pp. 102-111.
  27. J. M. Schmitt and K. Ben-Letaief, "Efficient Monte Carlo simulation of confocal microscopy in biological tissue," J. Opt. Soc. Am. A 13, 952-961 (1996). [CrossRef]
  28. C. F. Bohren and D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley InterScience, 1983).
  29. S. Bartel and A. H. Hielscher, "Monte Carlo simulations of the diffuse backscattering Mueller matrix for highly scattering media," Appl. Opt. 39, 1580-1588 (2000). [CrossRef]
  30. X. Wang, L. V. Wang, C.-W. Sun, and C.-C. Yang, "Polarized light propagation through scattering media: time-resolved Monte Carlo simulations and experiments," J. Biomed. Opt. 8, 608-617 (2003). [CrossRef] [PubMed]
  31. X. Wang and L. V. Wang, "Propagation of polarized light in birefringent turbid media: a Monte Carlo study," J. Biomed. Opt. 7, 279-290 (2002). [CrossRef] [PubMed]
  32. J. Wu, M. S. Feld, and R. P. Rava, "Analytical model for extracting intrinsic fluorescence in turbid media," Appl. Opt. 32, 3585-3595 (1993). [CrossRef] [PubMed]
  33. T. Tao, "Time-dependent fluorescence depolarization and Brownian rotational diffusion coefficients of macromolecules," Biopolymers 8, 609-632 (1969). [CrossRef]
  34. D. J. Griffiths, Introduction to Electrodynamics (Prentice-Hall, 1989).
  35. F. C. MacKintosh, J. X. Zhu, D. J. Pine, and D. A. Weitz, "Polarization memory of multiply scattered light," Phys. Rev. B 40, 9342-9345 (1989). [CrossRef]
  36. M. Gu, S. P. Schilders, and X. Gan, "Two-photon fluorescence imaging of microspheres embedded in turbid media," J. Mod. Opt. 47, 959-965 (2000). [CrossRef]
  37. C. R. Cantor and P. R. Schimmel, Biophysical Chemistry. Part II: Techniques for the Study of Biological Structure and Function (Freeman, 1980).
  38. S. A. Allison and J. M. Schurr, "Torsion dynamics and depolarization of fluorescence of linear macromolecules. I. Theory and application to DNA," Chem. Phys. 41, 35-59 (1979). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited