Abstract

This paper compares tandem scanning reflected light confocal microscopy and multi-photon excitation microscopy for the observation of human skin in vivo. Tandem scanning confocal light microscopy based on a white light source can provide video-rate image acquisition from the skin surface to the epidermal-dermal junction. Multi-photon excitation is induced by a 80 MHz pulse train of femtosecond laser pulses at 780 nm wave length. This nonlinear microscopic technique is inherently suitable for deep tissue fluorescence imaging. The relative merits of these two techniques can be identified by comparing movies of optical sections obtained from the forearm skin of the same volunteer.

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References

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  1. B. R. Masters, P. T. C. So, and E. Gratton, "Optical biopsy of in vivo human skin: multiphoton excitation microscopy," Lasers Med. Sci. 13,196-203 (1998).
    [CrossRef]
  2. C. Buehler, K. H. Kim, C. Y. Dong, B. R. Masters and P. T. C. So, "Innovations in two-photon deep tissue microscopy," IEEE Engineering in Medicine and Biology 18, 23-29 (1999).
    [CrossRef]
  3. B. R. Masters, P. T. C. So, K. H. Kim, C. Buehler, and E. Gratton, "Multiphoton excitation microscopy, confocal microscopy, and spectroscopy of living cells and tissues: functional metabolic imaging of human skin in vivo," in Methods in Enzymology, Confocal Microscopy, vol. 307, P. M. Conn, ed. (Academic Press, New York, 1999) Chap. 29.
    [CrossRef]
  4. P. Corcuff, G. Gonnord, G. E. Pierard, and J. L. Leveque, "In vivo confocal microscopy of human skin: a new design for cosmetology and dermatology," Scanning 18, 351-355 (1996).
    [CrossRef] [PubMed]
  5. P. Corcuff and J. L. Leveque, "In vivo vision of the human skin with the tandem scanning microscope," Dermatology 186, 50-54 (1993).
    [CrossRef] [PubMed]
  6. P. Corcuff, C. Bertrand and J. L. Leveque, "Morphometry of human epidermis in vivo by real-time confocal microscopy," Arch Dermatol Res. 285, 475-481(1993).
    [CrossRef] [PubMed]
  7. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb and R. Anderson, "In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast," J. Invest. Dermatol. 104, 946-952 (1995).
    [CrossRef] [PubMed]
  8. B. R. Masters, "Three-dimensional confocal microscopy of human skin in vivo: autofluorescence of normal skin," Bioimages 4, 13-19 (1996).
  9. B. R. Masters, D. J. Aziz, A. F. Gmitro, J. H. Kerr, T. C. O'Grady and L. Goldman, "Rapid observation of unfixed, unstained, human skin biopsy specimens with confocal microscopy and visualization," J. Biomed. Opt. 2, 437-445 (1997).
    [CrossRef]
  10. B. R. Masters, G. Gonnord and P. Corcuff, "Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope," J. Micros. 185, 329-338 (1997).
    [CrossRef]
  11. C. Bertrand and P. Corcuff, "In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy," Scanning 16, 150-154 (1994).
    [CrossRef] [PubMed]
  12. B. R. Masters and P. T. C. So, "Multi-photon excitation microscopy and confocal microscopy imaging of in vivo human skin: a comparison," Micros. Microanal. 5, 282-289 (1999).
  13. R. R. Anderson and J. A. Parrish, "The optics of human skin," J. Invest. Dermatol. 77,13-19 (1981).
    [CrossRef] [PubMed]
  14. W. J. Denk, J. P. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  15. W. J. Denk, D. W. Piston and W. W. Webb, Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum Press, New York, 1995).
  16. W. Denk, "Two-photon excitation in functional biological imaging," J. Biomed. Opt. 1, 296-304 (1996).
    [CrossRef]
  17. W. J. Denk, J. P. Strickler and W. W. Webb, "Two-photon laser microscopy," United States Patent, 5,034,613, July 23, 1991.
  18. P. T. C. So, C. Y. Chen, B. R. Masters, and K. M. Berland, "Two-photon excitation fluorescence microscopy," in Ann. Rev. Biomedical Engineering, M.L. Yarmush, K. R. Diller, M. Toner ed. (Annual Reviews, Palo Alto, CA 2000).
  19. K. Konig, P. T. C. So , W. W. Mantulin and E. Gratton, "Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes," Opt. Lett. 22, 135-136 (1997).
    [CrossRef] [PubMed]
  20. K. Konig, P. T. C. So , W. W. Mantulin, B. J. Tromberg and E. Gratton, "Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress," J. Micros. 183, 197-204(1996).
  21. T. Wilson, Confocal Microscopy, (Academic Press, London 1990).
  22. M. Gu, Principles of three-dimensional imaging in confocal microscopy, (World Scientific, Singapore 1996).
  23. T. R. Corle and G. S. Kino, Confocal scanning optical microscopy and related imaging systems (Academic Press, San Diego 1996).
  24. J. B. Pawley, Handbook of Biological Confocal Microscopy, (Plenum Press, New York, 1995).
    [CrossRef]
  25. B. R. Masters, Selected Papers on Confocal Microscopy, (SPIE Press, Bellingham, WA, 1996).
  26. P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong and E. Gratton, "Time-resolved fluorescence microscopy using two-photon excitation," Bioimaging 3, 49-63 (1995).
    [CrossRef]
  27. P. T. C. So, H. Kim, I. E. Kochevar, "Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures," Opt. Express 3, 339-350 (1998). http://www.opticsexpress.org/oearchive/source/6200.htm
    [CrossRef] [PubMed]
  28. B . Chance, "Pyridine nucleotide as an indicator of the oxygen requirements for energy-linked functions of mitochondria," Circ. Res. Suppl. 1, 38,I-31 - I-38 (1976).
  29. D. W. Piston, B. R. Masters and W. W. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Micros. 178, 20-27 (1995).
    [CrossRef]
  30. K-H. Kim, C. Buehler, and P. T. C. So, "High-speed two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
    [CrossRef]
  31. J. Bewersdorf, R. Pick and S. W. Hell, "Mulitfocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
    [CrossRef]
  32. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade and B. Athey "Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system," J. Microsc. 181, 253-259 (1996).
    [CrossRef] [PubMed]

Other

B. R. Masters, P. T. C. So, and E. Gratton, "Optical biopsy of in vivo human skin: multiphoton excitation microscopy," Lasers Med. Sci. 13,196-203 (1998).
[CrossRef]

C. Buehler, K. H. Kim, C. Y. Dong, B. R. Masters and P. T. C. So, "Innovations in two-photon deep tissue microscopy," IEEE Engineering in Medicine and Biology 18, 23-29 (1999).
[CrossRef]

B. R. Masters, P. T. C. So, K. H. Kim, C. Buehler, and E. Gratton, "Multiphoton excitation microscopy, confocal microscopy, and spectroscopy of living cells and tissues: functional metabolic imaging of human skin in vivo," in Methods in Enzymology, Confocal Microscopy, vol. 307, P. M. Conn, ed. (Academic Press, New York, 1999) Chap. 29.
[CrossRef]

P. Corcuff, G. Gonnord, G. E. Pierard, and J. L. Leveque, "In vivo confocal microscopy of human skin: a new design for cosmetology and dermatology," Scanning 18, 351-355 (1996).
[CrossRef] [PubMed]

P. Corcuff and J. L. Leveque, "In vivo vision of the human skin with the tandem scanning microscope," Dermatology 186, 50-54 (1993).
[CrossRef] [PubMed]

P. Corcuff, C. Bertrand and J. L. Leveque, "Morphometry of human epidermis in vivo by real-time confocal microscopy," Arch Dermatol Res. 285, 475-481(1993).
[CrossRef] [PubMed]

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb and R. Anderson, "In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast," J. Invest. Dermatol. 104, 946-952 (1995).
[CrossRef] [PubMed]

B. R. Masters, "Three-dimensional confocal microscopy of human skin in vivo: autofluorescence of normal skin," Bioimages 4, 13-19 (1996).

B. R. Masters, D. J. Aziz, A. F. Gmitro, J. H. Kerr, T. C. O'Grady and L. Goldman, "Rapid observation of unfixed, unstained, human skin biopsy specimens with confocal microscopy and visualization," J. Biomed. Opt. 2, 437-445 (1997).
[CrossRef]

B. R. Masters, G. Gonnord and P. Corcuff, "Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope," J. Micros. 185, 329-338 (1997).
[CrossRef]

C. Bertrand and P. Corcuff, "In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy," Scanning 16, 150-154 (1994).
[CrossRef] [PubMed]

B. R. Masters and P. T. C. So, "Multi-photon excitation microscopy and confocal microscopy imaging of in vivo human skin: a comparison," Micros. Microanal. 5, 282-289 (1999).

R. R. Anderson and J. A. Parrish, "The optics of human skin," J. Invest. Dermatol. 77,13-19 (1981).
[CrossRef] [PubMed]

W. J. Denk, J. P. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

W. J. Denk, D. W. Piston and W. W. Webb, Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum Press, New York, 1995).

W. Denk, "Two-photon excitation in functional biological imaging," J. Biomed. Opt. 1, 296-304 (1996).
[CrossRef]

W. J. Denk, J. P. Strickler and W. W. Webb, "Two-photon laser microscopy," United States Patent, 5,034,613, July 23, 1991.

P. T. C. So, C. Y. Chen, B. R. Masters, and K. M. Berland, "Two-photon excitation fluorescence microscopy," in Ann. Rev. Biomedical Engineering, M.L. Yarmush, K. R. Diller, M. Toner ed. (Annual Reviews, Palo Alto, CA 2000).

K. Konig, P. T. C. So , W. W. Mantulin and E. Gratton, "Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes," Opt. Lett. 22, 135-136 (1997).
[CrossRef] [PubMed]

K. Konig, P. T. C. So , W. W. Mantulin, B. J. Tromberg and E. Gratton, "Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress," J. Micros. 183, 197-204(1996).

T. Wilson, Confocal Microscopy, (Academic Press, London 1990).

M. Gu, Principles of three-dimensional imaging in confocal microscopy, (World Scientific, Singapore 1996).

T. R. Corle and G. S. Kino, Confocal scanning optical microscopy and related imaging systems (Academic Press, San Diego 1996).

J. B. Pawley, Handbook of Biological Confocal Microscopy, (Plenum Press, New York, 1995).
[CrossRef]

B. R. Masters, Selected Papers on Confocal Microscopy, (SPIE Press, Bellingham, WA, 1996).

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong and E. Gratton, "Time-resolved fluorescence microscopy using two-photon excitation," Bioimaging 3, 49-63 (1995).
[CrossRef]

P. T. C. So, H. Kim, I. E. Kochevar, "Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures," Opt. Express 3, 339-350 (1998). http://www.opticsexpress.org/oearchive/source/6200.htm
[CrossRef] [PubMed]

B . Chance, "Pyridine nucleotide as an indicator of the oxygen requirements for energy-linked functions of mitochondria," Circ. Res. Suppl. 1, 38,I-31 - I-38 (1976).

D. W. Piston, B. R. Masters and W. W. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Micros. 178, 20-27 (1995).
[CrossRef]

K-H. Kim, C. Buehler, and P. T. C. So, "High-speed two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
[CrossRef]

J. Bewersdorf, R. Pick and S. W. Hell, "Mulitfocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
[CrossRef]

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, W. H. Wade and B. Athey "Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system," J. Microsc. 181, 253-259 (1996).
[CrossRef] [PubMed]

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Figures (5)

Fig. 1.
Fig. 1.

Schematic drawing of a vertical section of human skin. The following layers are shown: stratum corneum, stratum granulosum, stratum spinosum, the basal layer, and the papillary dermis.

Fig. 2.
Fig. 2.

Schematic of a two-photon deep tissue microscope.

Fig. 3.
Fig. 3.

Frontal sections of human skin in vivo acquired with reflected light confocal microscopy (a-d) and acquired from multi-photon excitation microscopy with excitation at 780 nm (e-h). Images were acquired from the volar surface of the forearm at similar positions with the two techniques described in the Methods section. Images were acquired at the regions: 10 microns below surface in stratum corneum (a and e), cells of stratum spinosum (b and f), cells of basal layer (c and g), and within the dermis (d and h). Scale bars show 50 microns.

Fig. 4.
Fig. 4.

(2.29 Mb) Movie of stacks of optical sections from human skin in vivo. Optical sections were acquired with reflected light confocal microscopy. Intensity is mapped into colors to enhance visualization, high intensities are yellow and red, low intensities are green and black. The horizontal length of this field is 175 µm.

Fig. 5.
Fig. 5.

(1.45 Mb) Movie of stacks of optical sections from human skin in vivo. Optical sections were acquired with multi-photon excitation microscopy with excitation at 780 nm. Intensity is mapped into colors to enhance visualization; high intensities are red and yellow, low intensities are dark red. Reconstruction shows the cells 10 microns below the surface within the stratum corneum, the cells of the basal layer, and structures within the dermis. The horizontal length of this field is 175 µm.

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