Abstract

Spatial distribution of two-photon-excited fluorescence (TPF) for dye beneath the surface of a highly scattering medium was investigated with picosecond laser pulses at 1064 nm. The active scattering media consisted of a suspension of polystyrene particles in a solution of Rhodamine 590 Tetrafluoroborate dye. With the increase of scattering strength of the medium, the location of the maximum TPF intensity was found to move closer to the surface away from the focal region; the intensity of TPF was not confined to the focal region as in the case of nonscattering medium but was more evenly distributed. The spatial resolution of nonlinear optical microscopy for probing a scattering medium is degraded. Taking into account the scattering of the medium qualitatively explains the observed TPF spatial distribution.

© 1999 Optical Society of America

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Errata

Jinpin Ying, Feng Liu, and R. R. Alfano, "Spatial distribution of two-photon-excited fluorescence in scattering media: erratum," Appl. Opt. 38, 2151-2151 (1999)
https://www.osapublishing.org/ao/abstract.cfm?uri=ao-38-10-2151

References

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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. 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]
  4. K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature (London) 385, 161–165 (1997).
    [CrossRef]
  5. W. Denk, “Two-photon excitation in functional biological imaging,” J. Biomed. Opt. 1, 296–304 (1996).
    [CrossRef] [PubMed]
  6. J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, New York, 1995).
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  8. 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]
  9. 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, 530–480 (1997).
    [CrossRef] [PubMed]
  10. Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.
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    [CrossRef]
  12. 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]

1997

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]

K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature (London) 385, 161–165 (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]

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, 530–480 (1997).
[CrossRef] [PubMed]

1996

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

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.

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]

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

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.

K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature (London) 385, 161–165 (1997).
[CrossRef]

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

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

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.

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]

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

Harris, D.

Ho, P. P.

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]

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

Kleinfeld, D.

K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature (London) 385, 161–165 (1997).
[CrossRef]

Liu, F.

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]

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

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, 530–480 (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]

Pawley, J. B.

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

Piston, D. W.

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]

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]

Savage, H.

Savage, H. E.

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

Schantz, S.

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]

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

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, 530–480 (1997).
[CrossRef] [PubMed]

Sheppard, C.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

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]

Strickler, J. H.

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

Svoboda, K.

K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature (London) 385, 161–165 (1997).
[CrossRef]

Tank, D. W.

K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature (London) 385, 161–165 (1997).
[CrossRef]

Teich, M. C.

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

Wang, Q. Z.

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

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, 530–480 (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, 530–480 (1997).
[CrossRef] [PubMed]

Wilson, T.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

Zhadin, N.

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

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, 530–480 (1997).
[CrossRef] [PubMed]

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. Biomed. Opt.

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

J. Microsc.

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]

Nature (London)

K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature (London) 385, 161–165 (1997).
[CrossRef]

Opt. Lett.

Science

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, 530–480 (1997).
[CrossRef] [PubMed]

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

Other

Y. Guo, F. Liu, Q. Z. Wang, H. E. Savage, N. Zhadin, P. P. Ho, S. Schantz, R. R. Alfano, “Nonlinear optical histological spectroscopy and imaging of biological tissues,” in Digest of Topical Meeting on Advances in Optical Imaging and Photon Migration (Optical Society of America, Washington, D.C., 1998), pp. 323–324.

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

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

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).

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Figures (6)

Fig. 1
Fig. 1

Experimental setup to measure the spatial distribution of the TPF. (a) Experimental system: ND, neutral density filter. (b) Schematic of beam-propagation geometry.

Fig. 2
Fig. 2

TPF images (192 × 165 pixels) for active scattering media with different scattering strength: In inverse millimeters, (a) μ s = 0.00; (b) μ s = 0.37; (c) μ s = 0.72; (d) μ s = 1.06 (e) μ s = 1.40; (f) μ s = 1.73. Pixel size is 14 µm. The whole field of view of the image is 2.69 × 2.31 mm2.

Fig. 3
Fig. 3

TPF intensity profiles along the optical axis of the input laser beam for a medium with different scattering strength. (a) Semilogarithmic plot of unnormalized and calibrated TPF intensity profiles. (b) Normalized TPF profiles in which maximum intensity was set to 255 counts. The numbers on the curves indicate the scattering coefficients (inverse millimeters) of media corresponding to Fig. 2(a)2(f). Note that the unit of horizontal axis is changed to millimeters from pixels.

Fig. 4
Fig. 4

Plots of the shift of the TPF peak intensity position as a function of the scattering coefficient of the medium. Square symbols represent the experimental data. The solid curve is a fit to the data of Eq. (3) with z 0 = 0.49 mm, which was estimated from the TPF profiles for the neat dye sample in Fig. 3(a).

Fig. 5
Fig. 5

(a) Semilogarithmic plot of unnormalized TPF intensity profiles. (b) Normalized TPF profiles along the optical axis computed with Eq. (2) under the experimental condition in which z 0 = 0.49 mm and z s = 2.31 mm.

Fig. 6
Fig. 6

Semilogarithmic plot of integrated TPF profiles [Eq. (5)] for different scattering coefficients. The focal point is at 200 µm inside the scattering media, and z 0 = 2 µm.

Equations (5)

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

Iρ, z=I0w0wz2 exp-2ρ2w2zexp-μsz+zs,
ITPF0, z  I2ρ=0, z=I021+z/z02-2×exp-2μsz+zs.
zmax=-ls+ls2-z021/2,
ITPF0, z  0 I2y=0, zdx=π/4 w0I02×1+z/z02-3/2exp-2μsz+zs.
0 ITPFρ, z2πρdρ  0 I2ρ, z2πρdρ=π/4 w02I021+z/z02-1 exp-2μsz+zs.

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