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.

© 2001 Optical Society of America

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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, NewYork, 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. Optics,  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]
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    [Crossref] [PubMed]
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  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]
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    [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, and M. Toner ed. (Annual Reviews, Palo Alto, CA2000).
  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).
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  22. M. Gu, Principles of three-dimensional imaging in confocal microscopy, (World Scientific, Singapore1996).
  23. T. R. Corle and G. S. Kino, Confocal scanning optical microscopy and related imaging systems (Academic Press, San Diego1996).
  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, and I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures.” Optics Express,  3, 339–350 (1998). http://www.opticsexpress.org
    [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]

1999 (3)

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 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).

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

1998 (3)

J. Bewersdorf, R. Pick, and S. W. Hell, “Mulitfocal multiphoton microscopy,” Opt. Lett. 23, 655–657 (1998).
[Crossref]

P. T. C. So, H. Kim, and I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures.” Optics Express,  3, 339–350 (1998). http://www.opticsexpress.org
[Crossref] [PubMed]

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]

1997 (3)

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. Optics,  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]

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]

1996 (5)

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).

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

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]

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]

W . Denk, “Two-photon excitation in functional biological imaging.” J. Biomed. Optics 1, 296–304(1996).
[Crossref]

1995 (3)

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]

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]

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]

1994 (1)

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]

1993 (2)

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]

1990 (1)

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

1981 (1)

R. R. Anderson and J. A. Parrish, “The optics of human skin.” J. Invest Dermatol 77,13–19 (1981).
[Crossref] [PubMed]

1976 (1)

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).

Anderson, R.

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]

Anderson, R. R.

R. R. Anderson and J. A. Parrish, “The optics of human skin.” J. Invest Dermatol 77,13–19 (1981).
[Crossref] [PubMed]

Athey, B.

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]

Aziz, D. J.

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. Optics,  2,437–445 (1997).
[Crossref]

Berland, K. M.

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, 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, and M. Toner ed. (Annual Reviews, Palo Alto, CA2000).

Bertrand, C.

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]

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]

Bewersdorf, J.

Bliton, A. C.

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]

Brakenhoff, G. J.

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]

Buehler, C.

K-H. Kim, C. Buehler, and P. T. C. So, “High-speed two-photon scanning microscope,” Appl. Opt. 38, 6004–6009 (1999).
[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, NewYork, 1999) Chap. 29.
[Crossref]

Chance, B .

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).

Chen, C. Y.

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, and M. Toner ed. (Annual Reviews, Palo Alto, CA2000).

Corcuff, P.

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]

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]

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]

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]

Corle, T. R.

T. R. Corle and G. S. Kino, Confocal scanning optical microscopy and related imaging systems (Academic Press, San Diego1996).

Denk, W .

W . Denk, “Two-photon excitation in functional biological imaging.” J. Biomed. Optics 1, 296–304(1996).
[Crossref]

Denk, W. J.

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

Dong, C. Y.

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]

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]

Esterowitz, D.

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]

French, T.

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]

Gmitro, A. F.

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. Optics,  2,437–445 (1997).
[Crossref]

Goldman, L.

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. Optics,  2,437–445 (1997).
[Crossref]

Gonnord, G.

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]

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]

Gratton, E.

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]

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).

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]

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, NewYork, 1999) Chap. 29.
[Crossref]

Grossman, M.

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]

Gu, M.

M. Gu, Principles of three-dimensional imaging in confocal microscopy, (World Scientific, Singapore1996).

Hell, S. W.

Kerr, J. H.

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. Optics,  2,437–445 (1997).
[Crossref]

Kim, H.

P. T. C. So, H. Kim, and I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures.” Optics Express,  3, 339–350 (1998). http://www.opticsexpress.org
[Crossref] [PubMed]

Kim, K. H.

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, NewYork, 1999) Chap. 29.
[Crossref]

Kim, K-H.

Kino, G. S.

T. R. Corle and G. S. Kino, Confocal scanning optical microscopy and related imaging systems (Academic Press, San Diego1996).

Kochevar, I. E.

P. T. C. So, H. Kim, and I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures.” Optics Express,  3, 339–350 (1998). http://www.opticsexpress.org
[Crossref] [PubMed]

Konig, K.

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).

Leveque, J. L.

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, 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]

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]

Mantulin, W. W.

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).

Masters, B. R.

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).

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, and E. Gratton, “Optical biopsy of in vivo human skin: multiphoton excitation microscopy,” Lasers Med Sci 13,196–203 (1998).
[Crossref]

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. Optics,  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]

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

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]

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, NewYork, 1999) Chap. 29.
[Crossref]

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

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, and M. Toner ed. (Annual Reviews, Palo Alto, CA2000).

Norris, T.

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]

O’Grady, T. C.

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. Optics,  2,437–445 (1997).
[Crossref]

Parrish, J. A.

R. R. Anderson and J. A. Parrish, “The optics of human skin.” J. Invest Dermatol 77,13–19 (1981).
[Crossref] [PubMed]

Pawley, J. B.

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

Pick, R.

Pierard, G. E.

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]

Piston, D. W.

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]

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

Rajadhyaksha, M.

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]

So, P. T. C.

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 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).

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

P. T. C. So, H. Kim, and I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures.” Optics Express,  3, 339–350 (1998). http://www.opticsexpress.org
[Crossref] [PubMed]

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]

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).

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, 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, and M. Toner ed. (Annual Reviews, Palo Alto, CA2000).

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, NewYork, 1999) Chap. 29.
[Crossref]

Squier, J.

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]

Strickler, J. P.

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

Tromberg, B. J.

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).

Wade, W. H.

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]

Webb, R. H.

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]

Webb, W. W.

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]

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

Wilson, T.

T. Wilson, Confocal Microscopy, (Academic Press, London1990).

Yu, W. M.

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]

Appl. Opt. (1)

Arch Dermatol Res. (1)

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]

Bioimages (1)

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

Bioimaging (1)

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]

Circ. Res. Suppl. (1)

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).

Dermatology (1)

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]

IEEE Engineering in Medicine and Biology (1)

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]

J Microsc. (1)

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]

J. Biomed. Optics (2)

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. Optics,  2,437–445 (1997).
[Crossref]

W . Denk, “Two-photon excitation in functional biological imaging.” J. Biomed. Optics 1, 296–304(1996).
[Crossref]

J. Invest Dermatol (2)

R. R. Anderson and J. A. Parrish, “The optics of human skin.” J. Invest Dermatol 77,13–19 (1981).
[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]

J. Micros. (3)

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]

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. 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).

Lasers Med Sci (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]

Micros. Microanal. (1)

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).

Opt. Lett. (2)

Optics Express (1)

P. T. C. So, H. Kim, and I. E. Kochevar, “Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures.” Optics Express,  3, 339–350 (1998). http://www.opticsexpress.org
[Crossref] [PubMed]

Scanning (2)

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]

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]

Science (1)

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

Other (9)

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. 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, and M. Toner ed. (Annual Reviews, Palo Alto, CA2000).

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, NewYork, 1999) Chap. 29.
[Crossref]

T. Wilson, Confocal Microscopy, (Academic Press, London1990).

M. Gu, Principles of three-dimensional imaging in confocal microscopy, (World Scientific, Singapore1996).

T. R. Corle and G. S. Kino, Confocal scanning optical microscopy and related imaging systems (Academic Press, San Diego1996).

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).

Supplementary Material (2)

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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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