Abstract

The intensity of two-photon excited fluorescence (TPF) generated by ultrashort laser pulses was measured as a function of the depth of a focal point inside highly scattering media. The purpose was to investigate the spatial location of TPF in a scattering medium. Owing to the scattering, the intensity of the incident beam as well as the generated TPF signal was attenuated exponentially as the focal point was scanned into the medium. As the scattering strength of the medium was increased, the TPF was not confined to the focal region and had a wider distribution. These observations show that the scattering will result in the degradation of the ability of optical depth sectioning of nonlinear optical scanning microscopy.

© 2000 Optical Society of America

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  1. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. D. W. Piston, B. R. Masters, 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. Microsc. 178, 20–27 (1995).
    [CrossRef] [PubMed]
  3. S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
    [CrossRef] [PubMed]
  4. B. R. Master, P. T. C. So, E. Gratton, “Multiphoton excitation fluorescence microscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
    [CrossRef]
  5. Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, R. R. Alfano, “Second harmonic tomography of tissues,” Opt. Lett. 22, 1323–1325 (1997).
    [CrossRef]
  6. D. W. Piston, “Imaging living cells and tissues by two-photon excitation microscopy,” Trends Cell Biol. 9, 66–69 (1999).
    [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. A. Diaspro, M. Robello, “Multiphoton excitation microscopy to study biosystems,” Microsc. Anal. Americas Ed., Issue 35, 11–13 (March1999).
  9. R. R. Alfano, A. Katz, “Fluorescence and Raman spectroscopy for tissue diagnosis and characterization,” in Analytical Use of Fluorescent Probes in Oncology, E. Kohen, J. G. Hirschberg, eds. (Plenum, New York, 1997), pp. 81–89.
  10. W. F. Cheong, S. A. Prahl, A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
    [CrossRef]
  11. C. M. Blanca, C. Saloma, “Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998).
    [CrossRef]
  12. H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981), Chap. 9.
  13. B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991), Chap. 3, pp. 80–92.
    [CrossRef]
  14. M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
    [CrossRef] [PubMed]
  15. S. Lindek, E. H. K. Stelzer, S. W. Hell, “Two new high-resolution confocal fluorescence microscopes (4pi, theta) with one and two-photon excitation,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 417–430.
    [CrossRef]

1999

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

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

A. Diaspro, M. Robello, “Multiphoton excitation microscopy to study biosystems,” Microsc. Anal. Americas Ed., Issue 35, 11–13 (March1999).

1998

C. M. Blanca, C. Saloma, “Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium,” Appl. Opt. 37, 8092–8102 (1998).
[CrossRef]

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

1997

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

B. R. Master, P. T. C. So, E. Gratton, “Multiphoton excitation fluorescence microscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef]

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, R. R. Alfano, “Second harmonic tomography of tissues,” Opt. Lett. 22, 1323–1325 (1997).
[CrossRef]

1995

D. W. Piston, B. R. Masters, 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. Microsc. 178, 20–27 (1995).
[CrossRef] [PubMed]

1990

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

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

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, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, R. R. Alfano, “Second harmonic tomography of tissues,” Opt. Lett. 22, 1323–1325 (1997).
[CrossRef]

R. R. Alfano, A. Katz, “Fluorescence and Raman spectroscopy for tissue diagnosis and characterization,” in Analytical Use of Fluorescent Probes in Oncology, E. Kohen, J. G. Hirschberg, eds. (Plenum, New York, 1997), pp. 81–89.

Blanca, C. M.

Cheong, W. F.

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

Denk, W.

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

Diaspro, A.

A. Diaspro, M. Robello, “Multiphoton excitation microscopy to study biosystems,” Microsc. Anal. Americas Ed., Issue 35, 11–13 (March1999).

Gratton, E.

B. R. Master, P. T. C. So, E. Gratton, “Multiphoton excitation fluorescence microscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef]

Guo, Y.

Harris, D.

Hell, S. W.

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

S. Lindek, E. H. K. Stelzer, S. W. Hell, “Two new high-resolution confocal fluorescence microscopes (4pi, theta) with one and two-photon excitation,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 417–430.
[CrossRef]

Ho, P. P.

Hofmann, U. G.

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

Katz, A.

R. R. Alfano, A. Katz, “Fluorescence and Raman spectroscopy for tissue diagnosis and characterization,” in Analytical Use of Fluorescent Probes in Oncology, E. Kohen, J. G. Hirschberg, eds. (Plenum, New York, 1997), pp. 81–89.

Lindek, S.

S. Lindek, E. H. K. Stelzer, S. W. Hell, “Two new high-resolution confocal fluorescence microscopes (4pi, theta) with one and two-photon excitation,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 417–430.
[CrossRef]

Liu, F.

Maiti, S.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

Master, B. R.

B. R. Master, P. T. C. So, E. Gratton, “Multiphoton excitation fluorescence microscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef]

Masters, B. R.

D. W. Piston, B. R. Masters, 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. Microsc. 178, 20–27 (1995).
[CrossRef] [PubMed]

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]

D. W. Piston, B. R. Masters, 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. Microsc. 178, 20–27 (1995).
[CrossRef] [PubMed]

Prahl, S. A.

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

Robello, M.

A. Diaspro, M. Robello, “Multiphoton excitation microscopy to study biosystems,” Microsc. Anal. Americas Ed., Issue 35, 11–13 (March1999).

Sacks, P.

Saleh, B. E. A.

B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991), Chap. 3, pp. 80–92.
[CrossRef]

Saloma, C.

Savage, H.

Schantz, S.

Schrader, M.

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

Shear, J. B.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

So, P. T. C.

B. R. Master, P. T. C. So, E. Gratton, “Multiphoton excitation fluorescence microscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef]

Stelzer, E. H. K.

S. Lindek, E. H. K. Stelzer, S. W. Hell, “Two new high-resolution confocal fluorescence microscopes (4pi, theta) with one and two-photon excitation,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 417–430.
[CrossRef]

Strickler, J. H.

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

Teich, M. C.

B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991), Chap. 3, pp. 80–92.
[CrossRef]

van de Hulst, H. C.

H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981), Chap. 9.

Webb, W. W.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, 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. Microsc. 178, 20–27 (1995).
[CrossRef] [PubMed]

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

Welch, A. J.

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

Williams, R. M.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

Ying, J.

Zipfel, W. R.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

B. R. Master, P. T. C. So, E. Gratton, “Multiphoton excitation fluorescence microscopy of in vivo human skin,” Biophys. J. 72, 2405–2412 (1997).
[CrossRef]

IEEE J. Quantum Electron.

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

J. Microsc.

M. Schrader, U. G. Hofmann, S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191, 135–140 (1998).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, 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. Microsc. 178, 20–27 (1995).
[CrossRef] [PubMed]

Microsc. Anal. Americas Ed.

A. Diaspro, M. Robello, “Multiphoton excitation microscopy to study biosystems,” Microsc. Anal. Americas Ed., Issue 35, 11–13 (March1999).

Opt. Lett.

Science

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

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 275, 530–532 (1997).
[CrossRef] [PubMed]

Trends Cell Biol.

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

Other

R. R. Alfano, A. Katz, “Fluorescence and Raman spectroscopy for tissue diagnosis and characterization,” in Analytical Use of Fluorescent Probes in Oncology, E. Kohen, J. G. Hirschberg, eds. (Plenum, New York, 1997), pp. 81–89.

S. Lindek, E. H. K. Stelzer, S. W. Hell, “Two new high-resolution confocal fluorescence microscopes (4pi, theta) with one and two-photon excitation,” in Handbook of Biological Confocal Microscopy, 2nd ed., J. B. Pawley, ed. (Plenum, New York, 1995), pp. 417–430.
[CrossRef]

H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981), Chap. 9.

B. E. A. Saleh, M. C. Teich, Fundamentals of Photonics (Wiley, New York, 1991), Chap. 3, pp. 80–92.
[CrossRef]

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Figures (5)

Fig. 1
Fig. 1

(a) Schematic diagram of the experimental setup. (b) Schematic of beam propagation geometry. The beam is incident from the left, and w 0 is the beam waist.

Fig. 2
Fig. 2

(a) Measured TPF depth intensity profiles as a function of focal depth position for scattering media with a 0.304-µm-diameter polystyrene sphere. The scattering coefficients at 810 nm are (1), 0; (2), 0.78; (3), 1.57; (4), 2.32; (5), 3.07; and (6), 3.70 mm-1. The scattering coefficient at 550-nm TPF emission is 2.68 times that at 810 nm. The volume concentrations of the polystyrene sphere scatterer are 0, 0.13%, 0.26%, 0.39%, 0.51%, and 0.63%. (b) Computed TPF depth intensity profiles when the scattering coefficients in Fig. 2(a) and Eq. (5) are used.

Fig. 3
Fig. 3

(a) Measured TPF intensity depth profiles as a function of the focal depth position for scattering media with a 1.07-µm-diameter polystyrene sphere. The scattering coefficients at 810 nm are (1), 0; (2), 0.63; (3), 0.83; (4), 1.25; (5), 2.50; and (6), 5.00 mm-1. The scattering coefficient at 550-nm TPF emission is 1.73 times that at 810 nm. The volume concentration of the polystyrene sphere scatterer is 0, 0.025%, 0.03%, 0.05%, 0.1%, and 0.2%. (b) Computed TPF depth intensity profiles when the scattering coefficients in Fig. 3(a) and Eq. (5) are used.

Fig. 4
Fig. 4

Difference percentage between the experimental and the theoretical TPF intensity profiles for three different scattering strengths of each media with (a) 0.304- and (b) 1.07-µm-diameter polystyrene spheres.

Fig. 5
Fig. 5

TPF depth profile for a 1.07-µm sphere scattering medium with an inserted glass slice 100 µm thick for different scattering coefficients. The scattering coefficients are (1), 0; (2), 0.63; (3), 0.83; and (4) 1.25 mm-1.

Tables (1)

Tables Icon

Table 1 Comparison of the Theoretical Decay Slope of the TPF Profile with Experimental Results for Two Kinds of Scattering Media

Equations (6)

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

Itpf   IgrGemr, r d3r,
Iexρ, z=I0w0wz2 exp-2ρ2w2zexp-μsexz,
Igρ, z  Iex2ρ, z=I02w0wz4 exp-4ρ2w2zexp-2μsexz.
Gemr, r=exp-μsem+μaem|z-z|,
Itpfzs  011+z-zs2/z02×exp-2μsex+μsem+μaemzdz.
ΔI%=Iexzs-IthzsIexzs×100%,

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