Abstract

We investigate the application of hyperosmotic optical clearing agents to improve the image contrast and penetration depth in two-photon microscopy of human dermis ex vivo. We show that the agents glycerol, propylene glycol, and glucose all convey significant improvements and we provide results on their dynamic behaviour and the reversibility of the effect. At suitable concentrations, such agents have the potential to be compatible with living tissue and may possibly enhance in-vivo deep-tissue imaging.

© 2005 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |

  1. W. F. Cheong, S. A. Prahl, and A. J. Welch, �??A review of the optical properties of biological tissues,�?? IEEE J. Quantum Electron. 26, 2166-2185 (1990)
    [CrossRef]
  2. J. A. Kiernan, Histological and Histochemical Methods, 3rd Edition, (Oxford University Press, New York), 1999
  3. H. Liu, B. Beauvoit, M. Kimura, and B. Chance, �??Dependence of tissue optical properties on solute-induced changes in refractive index and osmolarity,�?? J. Biomed. Opt. 1, 200-211 (1996)
    [CrossRef]
  4. V. V. Tuchin, I. L. Maksimova, D. A. Zimnyakov, I. L. Kon, A. H. Mavlutov, and A. A. Mishin, �??Light propagation in tissue with controlled optical properties,�?? J. Biomed. Opt. 2, 401-417 (1997)
    [CrossRef]
  5. G. Vargas, E. K. Chan, J. K. Barton, H. G. Rylander, and A. J. Welch, "Use of an agent to reduce scattering in skin" Lasers in Surg. and Med. 24, 133-141 (1999)
    [CrossRef]
  6. W. Denk, J. H. Strickler, and W. W. Webb, �??Two-photon laser scanning fluorescence microscope,�?? Science 248, 73-76 (1990)
    [CrossRef] [PubMed]
  7. A. K. Dunn, V. P. Wallace, M. Coleno, M. W. Berns, B. J. Tromberg, �??Influence of optical properties on two-photon fluorescence imaging in turbid samples,�?? Appl. Opt. 39, 1194-1201 (2000)
    [CrossRef]
  8. M. Gu, X. Gan, A. Kisteman, M. G. Xu, �??Comparison of penetration depth between two-photon excitation and single-photon excitation in imaging through turbid tissue media,�?? Appl. Phys. Lett. 77, 1551-1553 (2000)
    [CrossRef]
  9. E. Beaurepaire, M. Oheim, and J. Mertz, �??Ultra-deep two-photon fluorescence excitation in turbid media,�?? Opt. Commun. 188, 25-29 (2001)
    [CrossRef]
  10. B. R. Masters, P. T. C. So, and E. Gratton, �??Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,�?? Biophys. J. 72, 2405-2412 (1997)
    [CrossRef] [PubMed]
  11. B. R. Masters and P. T. C. So, �??Confocal microscopy and multi-photon excitation microscopy of human skin in vivo,�?? Opt. Express 8, 2-10 (2001)
    [CrossRef] [PubMed]
  12. K. König and I Riemann, �??High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,�?? J. Biomed. Opt. 8, 432-439 (2003)
    [CrossRef] [PubMed]
  13. Y. He and R. K. Wang, �??Dynamic optical clearing effect of tissue impregnated with hyperosmotic agents and studied with optical coherence tomography,�?? J. Biomed. Opt. 9, 200-206 (2004)
    [CrossRef] [PubMed]
  14. A. T. Yeh, B. Choi, J. S. Nelson, and B. J. Tromberg, �??Reversible dissociation of collagen in tissue,�?? J. Invest. Dermatol. 121, 1332-1335 (2003)
    [CrossRef] [PubMed]
  15. A. F. Zuluaga, R. Drezek, T. Collier, R. Lotan, M. Follen, and R. Richards-Kortum, �??Contrast agents for confocal microscopy: how simple chemicals affect confocal images of normal and cancer cells in suspension,�?? J. Biomed. Opt. 7, 398-403 (2002)
    [CrossRef] [PubMed]
  16. I. V. Meglinski, A. N. Bashkatov, E. A. Genina, D. Y. Churmakov, and V. V. Tuchin, �??The enhancement of confocal images of tissue at bulk optical immersion,�?? Laser Physics 13, 65-69 (2003)
  17. L. Sacconi, I. M. Tolic-Nørrelykke, R. Antolini, and F. S. Pavone, �??Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope,�?? J. Biomed. Opt. 10 (1), (2005)
    [CrossRef] [PubMed]
  18. X. Wang, T. E. Milner, M. C. Change, and J. S. Nelson, �??Group refractive index measurement of dry and hydrated type I collagen films using optical low-coherence reflectometry,�?? J. Biomed. Opt. 1, 212-216 (1996)
    [CrossRef]
  19. R. K. Wang, X. Xu, V. V. Tuchin, and J. B. Elder, �??Concurrent enhancement of imaging depth and contrast for optical coherence tomography by hyperosmotic agents,�?? J. Opt. Soc. Am. B 18, 948-953 (2001)
    [CrossRef]
  20. R. Barer, �??Refractometry and interferometry of living cells,�?? J. Opt. Soc. Am. 47, 545-556 (1957)
    [CrossRef] [PubMed]
  21. G. Vargas, K. F. Chan, S. L. Thomsen, A. J. Welch, �??Use of osmotically active agents to alter optical properties of tissue: Effects on the detected fluorescence signal measured through skin,�?? Lasers in Surg. and Med. 29, 213-220 (2001)
    [CrossRef]

Appl. Opt. (1)

Appl. Phys. Lett. (1)

M. Gu, X. Gan, A. Kisteman, M. G. Xu, �??Comparison of penetration depth between two-photon excitation and single-photon excitation in imaging through turbid tissue media,�?? Appl. Phys. Lett. 77, 1551-1553 (2000)
[CrossRef]

Biophys. J. (1)

B. R. Masters, P. T. C. So, and E. Gratton, �??Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,�?? Biophys. J. 72, 2405-2412 (1997)
[CrossRef] [PubMed]

IEEE J. Quantum Electron. (1)

W. F. Cheong, S. A. Prahl, and A. J. Welch, �??A review of the optical properties of biological tissues,�?? IEEE J. Quantum Electron. 26, 2166-2185 (1990)
[CrossRef]

J. Biomed. Opt. (7)

H. Liu, B. Beauvoit, M. Kimura, and B. Chance, �??Dependence of tissue optical properties on solute-induced changes in refractive index and osmolarity,�?? J. Biomed. Opt. 1, 200-211 (1996)
[CrossRef]

V. V. Tuchin, I. L. Maksimova, D. A. Zimnyakov, I. L. Kon, A. H. Mavlutov, and A. A. Mishin, �??Light propagation in tissue with controlled optical properties,�?? J. Biomed. Opt. 2, 401-417 (1997)
[CrossRef]

K. König and I Riemann, �??High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,�?? J. Biomed. Opt. 8, 432-439 (2003)
[CrossRef] [PubMed]

Y. He and R. K. Wang, �??Dynamic optical clearing effect of tissue impregnated with hyperosmotic agents and studied with optical coherence tomography,�?? J. Biomed. Opt. 9, 200-206 (2004)
[CrossRef] [PubMed]

A. F. Zuluaga, R. Drezek, T. Collier, R. Lotan, M. Follen, and R. Richards-Kortum, �??Contrast agents for confocal microscopy: how simple chemicals affect confocal images of normal and cancer cells in suspension,�?? J. Biomed. Opt. 7, 398-403 (2002)
[CrossRef] [PubMed]

L. Sacconi, I. M. Tolic-Nørrelykke, R. Antolini, and F. S. Pavone, �??Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope,�?? J. Biomed. Opt. 10 (1), (2005)
[CrossRef] [PubMed]

X. Wang, T. E. Milner, M. C. Change, and J. S. Nelson, �??Group refractive index measurement of dry and hydrated type I collagen films using optical low-coherence reflectometry,�?? J. Biomed. Opt. 1, 212-216 (1996)
[CrossRef]

J. Invest. Dermatol. (1)

A. T. Yeh, B. Choi, J. S. Nelson, and B. J. Tromberg, �??Reversible dissociation of collagen in tissue,�?? J. Invest. Dermatol. 121, 1332-1335 (2003)
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

J. Opt. Soc. Am. B (1)

Laser Physics (1)

I. V. Meglinski, A. N. Bashkatov, E. A. Genina, D. Y. Churmakov, and V. V. Tuchin, �??The enhancement of confocal images of tissue at bulk optical immersion,�?? Laser Physics 13, 65-69 (2003)

Lasers in Surg. and Med. (2)

G. Vargas, E. K. Chan, J. K. Barton, H. G. Rylander, and A. J. Welch, "Use of an agent to reduce scattering in skin" Lasers in Surg. and Med. 24, 133-141 (1999)
[CrossRef]

G. Vargas, K. F. Chan, S. L. Thomsen, A. J. Welch, �??Use of osmotically active agents to alter optical properties of tissue: Effects on the detected fluorescence signal measured through skin,�?? Lasers in Surg. and Med. 29, 213-220 (2001)
[CrossRef]

Opt. Commun. (1)

E. Beaurepaire, M. Oheim, and J. Mertz, �??Ultra-deep two-photon fluorescence excitation in turbid media,�?? Opt. Commun. 188, 25-29 (2001)
[CrossRef]

Opt. Express (1)

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, �??Two-photon laser scanning fluorescence microscope,�?? Science 248, 73-76 (1990)
[CrossRef] [PubMed]

Other (1)

J. A. Kiernan, Histological and Histochemical Methods, 3rd Edition, (Oxford University Press, New York), 1999

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (3)

Fig. 1.
Fig. 1.

(A) Image stacks for a skin tissue sample immersed in PBS (upper) and after immersion for 7 minutes in glycerol (lower). The corresponding total intensity, contrast and relative contrast are plotted in (B), (C) and (D), respectively.

Fig. 2.
Fig. 2.

(A) A time-lapse sequence of images at 60 µm depth in the skin sample, for immersion in glycerol at time t=0 indicated by the arrow (the unit of time is seconds); (B) Contrast versus incubation time.

Fig. 3.
Fig. 3.

Contrast versus incubation time and depth for immersion in (A) glycerol, (B) propylene glycol and (C) glucose, preceded and followed by application of PBS. In the right column the results obtained at 60 and 80 micron depths are presented on magnified scales.

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

Contrast = i , j = 1 N lines I ij I ij ,
RC = 100 Δ Contrast Contrast = 100 Contrast [ OCA ] - Contrast [ PBS ] Contrast [ PBS ] ,

Metrics