Abstract

We investigated the effects of handling and fixation processes on the two-photon fluorescence spectroscopy of endogenous fluorophors in mouse skeletal muscle. The skeletal muscle was handled in one of two ways: either sectioned without storage or sectioned following storage in a freezer. The two-photon fluorescence spectra measured for different storage or fixation periods show a differential among those samples that were stored in water or were fixed either in formalin or methanol. The spectroscopic results indicate that formalin was the least disruptive fixative, having only a weak effect on the two-photon fluorescence spectroscopy of muscle tissue, whereas methanol had a significant influence on one of the autofluorescence peaks. The two handling processes yielded similar spectral information, indicating no different effects between them.

© 2000 Optical Society of America

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References

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  1. J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, New York, 1995).
  2. X. F. Wang, B. Herman, Fluorescence Imaging Spectroscopy and Microscopy (Wiley, New York, 1996).
  3. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
  4. S. P. Schilders, M. Gu, “Three-dimensional autofluorescence spectroscopy of rat skeletal muscle tissue under two-photon excitation,” Appl. Opt. 38, 720–723 (1999).
    [CrossRef]
  5. Y. Guo, Q. Z. Wang, P. P. Ho, N. Zhadin, F. Liu, S. Demos, D. Calistru, A. Tirksliunas, A. Katz, Y. Budansky, R. R. Alfano, “Two-photon excitation of fluorescence from chicken tissue,” Appl. Opt. 36, 968–970 (1997).
    [CrossRef] [PubMed]
  6. A. Lago, A. T. Obeidat, A. E. Kaplan, J. B. Khurgin, P. L. Shkolnikov, M. D. Stern, “Two-photon-induced fluorescence of biological markers based on optical fibers,” Opt. Lett. 20, 2054–2056 (1995).
    [CrossRef] [PubMed]
  7. J. Ying, F. Liu, R. R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt. 38, 224–229 (1999).
    [CrossRef]
  8. K. König, A. Ruck, H. Schneckenburger, “Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin,” Opt. Eng. 31, 1470–1474 (1992).
    [CrossRef]
  9. R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
    [CrossRef]
  10. R. R. Alfano, A. Pradhan, G. C. Tang, S. J. Wahl, “Optical spectroscopic diagnosis of cancer and normal breast tissues,” J. Opt. Soc. Am. B. 6, 1015–1023 (1989).
    [CrossRef]
  11. H. Schneckenburger, K. König, “Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators,” Opt. Eng. 31, 1447–1451 (1992).
    [CrossRef]
  12. H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
    [CrossRef]
  13. H. K. Seidlitz, K. Stettmaier, J. M. Wessels, H. Schneckenburger, “Intracellular fluorescence polarization, picosecond kinetics, and light-induced reactions of photosensitizing porphyrins,” Opt. Eng. 31, 1482–1486 (1992).
    [CrossRef]
  14. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
    [CrossRef]
  15. P. T. C. So, B. R. Masters, E. Gratton, I. E. Kochevar, “Two-photon optical biopsy of thick tissues,” Biomed. Opt. Spectrosc. Diagn. 22, 417–419 (1998).
  16. D. W. Piston, “Imaging living cells and tissues by two-photon excitation microscopy,” Trends Cell Biol. 9, 66–69 (1999).
    [CrossRef] [PubMed]
  17. B. R. Masters, P. T. C. So, W. Mantulin, E. Gratton, “Tissue microscopy and spectroscopy: a two-photon approach,” Biomed. Opt. Spectrosc. Diagn. 22, 420–422 (1998).
  18. 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.epubs.osa.org/opticsexpress .
  19. B. Chance, P. Cohen, F. Jobsis, B. Schoener, “Intracellular oxidation states in vivo,” Science 137(3529), 499–508 (1962).
  20. J. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 36–43 (1979).
    [CrossRef] [PubMed]
  21. R. C. Benson, R. A. Meyer, M. E. Zauba, G. M. McKhann, “Cellular autofluorescence—is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
    [CrossRef] [PubMed]
  22. R. Bacallao, K. Kiai, L. Jesaitis, “Guiding principles of specimen preservation for confocal fluorescence microscopy,” in Handbook of Biological Confocal Microscopy, 2nd. ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 311–323.

1999 (3)

1998 (3)

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.epubs.osa.org/opticsexpress .

B. R. Masters, P. T. C. So, W. Mantulin, E. Gratton, “Tissue microscopy and spectroscopy: a two-photon approach,” Biomed. Opt. Spectrosc. Diagn. 22, 420–422 (1998).

P. T. C. So, B. R. Masters, E. Gratton, I. E. Kochevar, “Two-photon optical biopsy of thick tissues,” Biomed. Opt. Spectrosc. Diagn. 22, 417–419 (1998).

1997 (1)

1995 (2)

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
[CrossRef]

A. Lago, A. T. Obeidat, A. E. Kaplan, J. B. Khurgin, P. L. Shkolnikov, M. D. Stern, “Two-photon-induced fluorescence of biological markers based on optical fibers,” Opt. Lett. 20, 2054–2056 (1995).
[CrossRef] [PubMed]

1992 (4)

K. König, A. Ruck, H. Schneckenburger, “Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin,” Opt. Eng. 31, 1470–1474 (1992).
[CrossRef]

H. Schneckenburger, K. König, “Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators,” Opt. Eng. 31, 1447–1451 (1992).
[CrossRef]

H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
[CrossRef]

H. K. Seidlitz, K. Stettmaier, J. M. Wessels, H. Schneckenburger, “Intracellular fluorescence polarization, picosecond kinetics, and light-induced reactions of photosensitizing porphyrins,” Opt. Eng. 31, 1482–1486 (1992).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).

1989 (1)

R. R. Alfano, A. Pradhan, G. C. Tang, S. J. Wahl, “Optical spectroscopic diagnosis of cancer and normal breast tissues,” J. Opt. Soc. Am. B. 6, 1015–1023 (1989).
[CrossRef]

1987 (1)

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

1979 (2)

J. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 36–43 (1979).
[CrossRef] [PubMed]

R. C. Benson, R. A. Meyer, M. E. Zauba, G. M. McKhann, “Cellular autofluorescence—is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
[CrossRef] [PubMed]

1962 (1)

B. Chance, P. Cohen, F. Jobsis, B. Schoener, “Intracellular oxidation states in vivo,” Science 137(3529), 499–508 (1962).

Alfano, R. R.

J. Ying, F. Liu, R. R. Alfano, “Spatial distribution of two-photon-excited fluorescence in scattering media,” Appl. Opt. 38, 224–229 (1999).
[CrossRef]

Y. Guo, Q. Z. Wang, P. P. Ho, N. Zhadin, F. Liu, S. Demos, D. Calistru, A. Tirksliunas, A. Katz, Y. Budansky, R. R. Alfano, “Two-photon excitation of fluorescence from chicken tissue,” Appl. Opt. 36, 968–970 (1997).
[CrossRef] [PubMed]

R. R. Alfano, A. Pradhan, G. C. Tang, S. J. Wahl, “Optical spectroscopic diagnosis of cancer and normal breast tissues,” J. Opt. Soc. Am. B. 6, 1015–1023 (1989).
[CrossRef]

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

Anderson, R. R.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
[CrossRef]

Aubin, J.

J. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 36–43 (1979).
[CrossRef] [PubMed]

Bacallao, R.

R. Bacallao, K. Kiai, L. Jesaitis, “Guiding principles of specimen preservation for confocal fluorescence microscopy,” in Handbook of Biological Confocal Microscopy, 2nd. ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 311–323.

Benson, R. C.

R. C. Benson, R. A. Meyer, M. E. Zauba, G. M. McKhann, “Cellular autofluorescence—is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
[CrossRef] [PubMed]

Budansky, Y.

Calistru, D.

Chance, B.

B. Chance, P. Cohen, F. Jobsis, B. Schoener, “Intracellular oxidation states in vivo,” Science 137(3529), 499–508 (1962).

Choy, D. S. J.

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

Cohen, P.

B. Chance, P. Cohen, F. Jobsis, B. Schoener, “Intracellular oxidation states in vivo,” Science 137(3529), 499–508 (1962).

Demos, S.

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).

Esterowitz, D.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
[CrossRef]

Gratton, E.

P. T. C. So, B. R. Masters, E. Gratton, I. E. Kochevar, “Two-photon optical biopsy of thick tissues,” Biomed. Opt. Spectrosc. Diagn. 22, 417–419 (1998).

B. R. Masters, P. T. C. So, W. Mantulin, E. Gratton, “Tissue microscopy and spectroscopy: a two-photon approach,” Biomed. Opt. Spectrosc. Diagn. 22, 420–422 (1998).

Grossman, M.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
[CrossRef]

Gu, M.

Guo, Y.

Herman, B.

X. F. Wang, B. Herman, Fluorescence Imaging Spectroscopy and Microscopy (Wiley, New York, 1996).

Ho, P. P.

Jesaitis, L.

R. Bacallao, K. Kiai, L. Jesaitis, “Guiding principles of specimen preservation for confocal fluorescence microscopy,” in Handbook of Biological Confocal Microscopy, 2nd. ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 311–323.

Jobsis, F.

B. Chance, P. Cohen, F. Jobsis, B. Schoener, “Intracellular oxidation states in vivo,” Science 137(3529), 499–508 (1962).

Kaplan, A. E.

Katz, A.

Khurgin, J. B.

Kiai, K.

R. Bacallao, K. Kiai, L. Jesaitis, “Guiding principles of specimen preservation for confocal fluorescence microscopy,” in Handbook of Biological Confocal Microscopy, 2nd. ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 311–323.

Kim, H.

Kochevar, I. E.

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.epubs.osa.org/opticsexpress .

P. T. C. So, B. R. Masters, E. Gratton, I. E. Kochevar, “Two-photon optical biopsy of thick tissues,” Biomed. Opt. Spectrosc. Diagn. 22, 417–419 (1998).

König, K.

K. König, A. Ruck, H. Schneckenburger, “Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin,” Opt. Eng. 31, 1470–1474 (1992).
[CrossRef]

H. Schneckenburger, K. König, “Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators,” Opt. Eng. 31, 1447–1451 (1992).
[CrossRef]

Lago, A.

Lam, W.

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

Liu, F.

Mantulin, W.

B. R. Masters, P. T. C. So, W. Mantulin, E. Gratton, “Tissue microscopy and spectroscopy: a two-photon approach,” Biomed. Opt. Spectrosc. Diagn. 22, 420–422 (1998).

Masters, B. R.

B. R. Masters, P. T. C. So, W. Mantulin, E. Gratton, “Tissue microscopy and spectroscopy: a two-photon approach,” Biomed. Opt. Spectrosc. Diagn. 22, 420–422 (1998).

P. T. C. So, B. R. Masters, E. Gratton, I. E. Kochevar, “Two-photon optical biopsy of thick tissues,” Biomed. Opt. Spectrosc. Diagn. 22, 417–419 (1998).

McKhann, G. M.

R. C. Benson, R. A. Meyer, M. E. Zauba, G. M. McKhann, “Cellular autofluorescence—is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
[CrossRef] [PubMed]

Meyer, R. A.

R. C. Benson, R. A. Meyer, M. E. Zauba, G. M. McKhann, “Cellular autofluorescence—is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
[CrossRef] [PubMed]

Obeidat, A. T.

Opher, E.

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

Pawley, J. B.

J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, New York, 1995).

Piston, D. W.

D. W. Piston, “Imaging living cells and tissues by two-photon excitation microscopy,” Trends Cell Biol. 9, 66–69 (1999).
[CrossRef] [PubMed]

Pradhan, A.

R. R. Alfano, A. Pradhan, G. C. Tang, S. J. Wahl, “Optical spectroscopic diagnosis of cancer and normal breast tissues,” J. Opt. Soc. Am. B. 6, 1015–1023 (1989).
[CrossRef]

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

Rajadhyaksha, M.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
[CrossRef]

Ruck, A.

H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
[CrossRef]

K. König, A. Ruck, H. Schneckenburger, “Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin,” Opt. Eng. 31, 1470–1474 (1992).
[CrossRef]

Schilders, S. P.

Schneckenburger, H.

K. König, A. Ruck, H. Schneckenburger, “Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin,” Opt. Eng. 31, 1470–1474 (1992).
[CrossRef]

H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
[CrossRef]

H. Schneckenburger, K. König, “Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators,” Opt. Eng. 31, 1447–1451 (1992).
[CrossRef]

H. K. Seidlitz, K. Stettmaier, J. M. Wessels, H. Schneckenburger, “Intracellular fluorescence polarization, picosecond kinetics, and light-induced reactions of photosensitizing porphyrins,” Opt. Eng. 31, 1482–1486 (1992).
[CrossRef]

Schoener, B.

B. Chance, P. Cohen, F. Jobsis, B. Schoener, “Intracellular oxidation states in vivo,” Science 137(3529), 499–508 (1962).

Seidlitz, H. K.

H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
[CrossRef]

H. K. Seidlitz, K. Stettmaier, J. M. Wessels, H. Schneckenburger, “Intracellular fluorescence polarization, picosecond kinetics, and light-induced reactions of photosensitizing porphyrins,” Opt. Eng. 31, 1482–1486 (1992).
[CrossRef]

Shkolnikov, P. L.

So, P. T. C.

B. R. Masters, P. T. C. So, W. Mantulin, E. Gratton, “Tissue microscopy and spectroscopy: a two-photon approach,” Biomed. Opt. Spectrosc. Diagn. 22, 420–422 (1998).

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.epubs.osa.org/opticsexpress .

P. T. C. So, B. R. Masters, E. Gratton, I. E. Kochevar, “Two-photon optical biopsy of thick tissues,” Biomed. Opt. Spectrosc. Diagn. 22, 417–419 (1998).

Stern, M. D.

Stettmaier, K.

H. K. Seidlitz, K. Stettmaier, J. M. Wessels, H. Schneckenburger, “Intracellular fluorescence polarization, picosecond kinetics, and light-induced reactions of photosensitizing porphyrins,” Opt. Eng. 31, 1482–1486 (1992).
[CrossRef]

Straub, W.

H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).

Tang, G. C.

R. R. Alfano, A. Pradhan, G. C. Tang, S. J. Wahl, “Optical spectroscopic diagnosis of cancer and normal breast tissues,” J. Opt. Soc. Am. B. 6, 1015–1023 (1989).
[CrossRef]

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

Tirksliunas, A.

Wahl, S. J.

R. R. Alfano, A. Pradhan, G. C. Tang, S. J. Wahl, “Optical spectroscopic diagnosis of cancer and normal breast tissues,” J. Opt. Soc. Am. B. 6, 1015–1023 (1989).
[CrossRef]

Wang, Q. Z.

Wang, X. F.

X. F. Wang, B. Herman, Fluorescence Imaging Spectroscopy and Microscopy (Wiley, New York, 1996).

Webb, R. H.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).

Wessels, J. M.

H. K. Seidlitz, K. Stettmaier, J. M. Wessels, H. Schneckenburger, “Intracellular fluorescence polarization, picosecond kinetics, and light-induced reactions of photosensitizing porphyrins,” Opt. Eng. 31, 1482–1486 (1992).
[CrossRef]

H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
[CrossRef]

Ying, J.

Zauba, M. E.

R. C. Benson, R. A. Meyer, M. E. Zauba, G. M. McKhann, “Cellular autofluorescence—is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
[CrossRef] [PubMed]

Zhadin, N.

Appl. Opt. (3)

Biomed. Opt. Spectrosc. Diagn. (2)

B. R. Masters, P. T. C. So, W. Mantulin, E. Gratton, “Tissue microscopy and spectroscopy: a two-photon approach,” Biomed. Opt. Spectrosc. Diagn. 22, 420–422 (1998).

P. T. C. So, B. R. Masters, E. Gratton, I. E. Kochevar, “Two-photon optical biopsy of thick tissues,” Biomed. Opt. Spectrosc. Diagn. 22, 417–419 (1998).

IEEE J. Quantum Electron. (1)

R. R. Alfano, G. C. Tang, A. Pradhan, W. Lam, D. S. J. Choy, E. Opher, “Fluorescence spectra from cancerous and normal human breast and lung tissues,” IEEE J. Quantum Electron. 23, 1806–1811 (1987).
[CrossRef]

J. Histochem. Cytochem. (2)

J. Aubin, “Autofluorescence of viable cultured mammalian cells,” J. Histochem. Cytochem. 27, 36–43 (1979).
[CrossRef] [PubMed]

R. C. Benson, R. A. Meyer, M. E. Zauba, G. M. McKhann, “Cellular autofluorescence—is it due to flavins?” J. Histochem. Cytochem. 27, 44–48 (1979).
[CrossRef] [PubMed]

J. Invest. Dermatol. (1)

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 380–386 (1995).
[CrossRef]

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

R. R. Alfano, A. Pradhan, G. C. Tang, S. J. Wahl, “Optical spectroscopic diagnosis of cancer and normal breast tissues,” J. Opt. Soc. Am. B. 6, 1015–1023 (1989).
[CrossRef]

Opt. Eng. (4)

H. Schneckenburger, K. König, “Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators,” Opt. Eng. 31, 1447–1451 (1992).
[CrossRef]

H. Schneckenburger, W. Straub, A. Ruck, H. K. Seidlitz, J. M. Wessels, “Microscopic fluorescence spectroscopy and diagnosis,” Opt. Eng. 31, 995–999 (1992).
[CrossRef]

H. K. Seidlitz, K. Stettmaier, J. M. Wessels, H. Schneckenburger, “Intracellular fluorescence polarization, picosecond kinetics, and light-induced reactions of photosensitizing porphyrins,” Opt. Eng. 31, 1482–1486 (1992).
[CrossRef]

K. König, A. Ruck, H. Schneckenburger, “Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin,” Opt. Eng. 31, 1470–1474 (1992).
[CrossRef]

Opt. Express (1)

Opt. Lett. (1)

Science (2)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).

B. Chance, P. Cohen, F. Jobsis, B. Schoener, “Intracellular oxidation states in vivo,” Science 137(3529), 499–508 (1962).

Trends Cell Biol. (1)

D. W. Piston, “Imaging living cells and tissues by two-photon excitation microscopy,” Trends Cell Biol. 9, 66–69 (1999).
[CrossRef] [PubMed]

Other (3)

J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, New York, 1995).

X. F. Wang, B. Herman, Fluorescence Imaging Spectroscopy and Microscopy (Wiley, New York, 1996).

R. Bacallao, K. Kiai, L. Jesaitis, “Guiding principles of specimen preservation for confocal fluorescence microscopy,” in Handbook of Biological Confocal Microscopy, 2nd. ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 311–323.

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Figures (7)

Fig. 1
Fig. 1

Schematic diagram of the two-photon fluorescence microscope. DM, dichroic mirror; M1 and M2, mirrors; PMT, photomultiplier tube.

Fig. 2
Fig. 2

Two-photon fluorescence (a) image and (b) spectrum of fresh mouse skeletal muscle. The squares marked A and B in (a) are the measured spots that correspond to the two similarly labeled spectra in (b).

Fig. 3
Fig. 3

Measured two-photon fluorescence intensity plotted as a function of the storage or the fixation period for mouse skeletal tissues that were stored in water or were fixed in formalin or methanol.

Fig. 4
Fig. 4

Fluorescence intensity plotted as a function of the excitation power measured at the fluorescence peak at the wavelength of 470 nm.

Fig. 5
Fig. 5

Measured two-photon fluorescence spectra for group 1 samples that were stored in water or were fixed in formalin or methanol for 1, 2, 3, or 4 days.

Fig. 6
Fig. 6

Measured two-photon fluorescence spectra for group 2 samples that were stored in water or were fixed in formalin or methanol for 1, 2, 3, or 4 days.

Fig. 7
Fig. 7

Measured two-photon fluorescence spectra for group 1 muscle stored in water for 5 or 6 days.

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