Abstract

Image contrast enhancement is investigated for two-photon excitation fluorescence images of a microscopic sample that is buried underneath a turbid medium. The image contrast, which deteriorates rapidly with sample depth because of scattering loss, is enhanced by an increase in the average excitation power of the focused Gaussian (the TEM00 mode) beam according to a compensation relation that has been derived by use of a Monte Carlo analysis of the scattering problem. A correct increase in the excitation power results in a detected fluorescence signal that remains invariant with sample depth. The scheme is demonstrated on images of DAPI-stained nuclei cells viewed underneath a suspension of 0.105-μm-diameter polystyrene spheres.

© 1998 Optical Society of America

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References

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  20. C = (1/2)gϕησ2Pk, where η is the fluorescence quantum efficiency of the dye, ϕ is the fluorescence collection efficiency of the measurement system, k is the dye concentration, and g = 〈Ie2(t)〉/〈Ie(t)〉2 is a measure of the temporal coherence of the excitation source.

1998

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Biol. 43, 1741–1759 (1998).
[CrossRef] [PubMed]

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

1997

1996

1995

1994

1990

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

L. Grossweiner, J. Karagiannes, P. Johnson, Z. Zhang, “Gaussian beam spread in biological tissues,” Appl. Opt. 29, 379–383 (1990).
[CrossRef] [PubMed]

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

Armas, M.

Ben-Letaief, K.

Berne, B.

B. Berne, R. Pecora, Dynamic Light Scattering with Applications to Chemistry, Biology, and Physics (Wiley, New York, 1976).

Blanca, C.

Cambaliza, M. O.

Cheng, H.

Cheong, W.

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

Delaney, K.

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

Denk, W.

W. Denk, K. Svoboda, “Photon upmanship: Why multiphoton is more than a gimmick,” Neuron 18, 351–357 (1997).
[CrossRef] [PubMed]

K. Svoboda, W. Denk, W. H. Knox, S. Tsuda, “Two-photon-excitation scanning microscopy of living neurons with a saturable Bragg reflector mode-locked diode-pumped Cr:LiSrAlFl laser,” Opt. Lett. 21, 1411–1413 (1996).
[CrossRef] [PubMed]

R. Yuste, W. Denk, “Dendritic spines as basic functional units of neuronal integration,” Nature (London) 375, 682–684 (1995).
[CrossRef]

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

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

Fisher, W.

Gelperin, A.

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

Grossweiner, L.

Johnson, P.

Kagiwada, H.

H. Kagiwada, R. Kalaba, S. Ueno, Multiple Scattering Processes: Inverse and Direct (Addison-Wesley, London, 1975).

Kalaba, R.

H. Kagiwada, R. Kalaba, S. Ueno, Multiple Scattering Processes: Inverse and Direct (Addison-Wesley, London, 1975).

Karagiannes, J.

Kirby, M. S.

Kleinfeld, D.

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

Knox, W. H.

Knuttel, A.

Kondoh, H.

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Biol. 43, 1741–1759 (1998).
[CrossRef] [PubMed]

Lederer, W.

Newton, R.

R. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).

Palmes-Saloma, C.

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Biol. 43, 1741–1759 (1998).
[CrossRef] [PubMed]

Pecora, R.

B. Berne, R. Pecora, Dynamic Light Scattering with Applications to Chemistry, Biology, and Physics (Wiley, New York, 1976).

Piston, D.

Potter, S.

S. Potter, “Vital imaging: two photons are better than one,” Curr. Biol. 6, 1595–1598 (1996).
[CrossRef] [PubMed]

Prahl, S.

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

Saloma, C.

Schmitt, J.

Seaton, C.

Strickler, J.

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

Strowbridge, B.

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

Svoboda, K.

Tank, D.

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

Tsuda, S.

Ueno, S.

H. Kagiwada, R. Kalaba, S. Ueno, Multiple Scattering Processes: Inverse and Direct (Addison-Wesley, London, 1975).

Wachter, E.

Webb, W.

Welch, A.

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

Xu, C.

Yadlowsky, M.

Yuste, R.

R. Yuste, W. Denk, “Dendritic spines as basic functional units of neuronal integration,” Nature (London) 375, 682–684 (1995).
[CrossRef]

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

Zhang, Z.

Appl. Opt.

Appl. Spectrosc.

Curr. Biol.

S. Potter, “Vital imaging: two photons are better than one,” Curr. Biol. 6, 1595–1598 (1996).
[CrossRef] [PubMed]

IEEE J. Quantum Electron.

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

J. Neurosci. Methods

W. Denk, K. Delaney, A. Gelperin, D. Kleinfeld, B. Strowbridge, D. Tank, R. Yuste, “Anatomical and functional imaging of neurons using two-photon laser scanning microscopy,” J. Neurosci. Methods 54, 151–162 (1994).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

J. Opt. Soc. Am. B

Nature (London)

R. Yuste, W. Denk, “Dendritic spines as basic functional units of neuronal integration,” Nature (London) 375, 682–684 (1995).
[CrossRef]

Neuron

W. Denk, K. Svoboda, “Photon upmanship: Why multiphoton is more than a gimmick,” Neuron 18, 351–357 (1997).
[CrossRef] [PubMed]

Opt. Lett.

Phys. Med. Biol.

C. Saloma, C. Palmes-Saloma, H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in turbid medium,” Phys. Med. Biol. 43, 1741–1759 (1998).
[CrossRef] [PubMed]

Science

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

Other

C = (1/2)gϕησ2Pk, where η is the fluorescence quantum efficiency of the dye, ϕ is the fluorescence collection efficiency of the measurement system, k is the dye concentration, and g = 〈Ie2(t)〉/〈Ie(t)〉2 is a measure of the temporal coherence of the excitation source.

B. Berne, R. Pecora, Dynamic Light Scattering with Applications to Chemistry, Biology, and Physics (Wiley, New York, 1976).

H. Kagiwada, R. Kalaba, S. Ueno, Multiple Scattering Processes: Inverse and Direct (Addison-Wesley, London, 1975).

R. Newton, Scattering Theory of Waves and Particles (McGraw-Hill, New York, 1966).

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Figures (8)

Fig. 1
Fig. 1

Optical setup for analyzing the behavior of 2PEF imaging in a turbid medium with MC techniques. Points S, S′, and S″ are conjugate points, and lenses L1, L2, and L3 are identical. (DM is the dichroic mirror.)

Fig. 2
Fig. 2

Plots of excitation photon fraction E versus h/ d s for the following scattering conditions: (a) anisotropic and (b) isotropic, where n = 1.59, λ e = 750 nm, and N e = 107. The solid lines are given by E = exp(-h/ d s ) and describe the attenuation of a 6-mm-diameter collimated beam. Different NA values of the focusing lens are considered.

Fig. 3
Fig. 3

Plots of fluorescence photon fraction F versus h/ d s for the following scattering conditions: (a) anisotropic and (b) isotropic, where n = 1.59, λ f = 750 nm, and N f = 107. Different NA values are considered where the radius of fluorescing spot w 0 = λ e /2nNA, where λ e = 750 nm and n = 1.59.

Fig. 4
Fig. 4

Plots of F versus h/ d s for the following scattering conditions: (a) anisotropic and (b) isotropic, where n = 1.59, NA = 1, λ f = 750 nm, and N f = 107. Different values of the photodetector radius p are considered.

Fig. 5
Fig. 5

Two-photon excitation fluorescence microscope setup. The collimated excitation beam is directed into the focusing objective lens (dry, NA = 0.5).

Fig. 6
Fig. 6

Optical setup of the scattering experiment. The scattering medium that consists of 0.0823-μm-diameter polystyrene latex spheres (index of refraction, n = 1.59) is suspended in distilled water. The axial position of the sample is adjusted with a z-translation stage.

Fig. 7
Fig. 7

Plots of 〈F〉 (cross hairs) against 〈P 0〉 for different h values. The dotted curves are theoretical as given by Eq. (2). The 〈P 0〉 values representing the intersections between a horizontal (constant 〈F〉) line and the h curves yield the same amount of fluorescence signal.

Fig. 8
Fig. 8

(a) Unscattered 2PEF image of DAPI-stained cell nuclei, (b) image of cell nuclei under a 955-μm-thick scattering medium (〈P 0〉 = 10.22 mW), and (c) contrast-compensated image obtained with 〈P 0〉 = 14.258 mW. Image size: 256 × 256 pixels.

Tables (2)

Tables Icon

Table 1 Values of α e and E0 as a Function of NA Value

Tables Icon

Table 2 Values of Polynomial Coefficientsa

Equations (11)

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α e NA = 3.27 × 10 - 2 NA 2 + 1.89 × 10 - 2 NA + 1.001 .
α e NA = 3.42 × 10 - 2 NA 2 + 2.08 × 10 - 2 NA + 1.002 .
F h / d s = a 4 h / d s 4 + a 3 h / d s 3 + a 2 h / d s 2 + a 1 h / d s + a 0 ,
F e C P 0 E 0   exp - α e h / d s 2 P 2 = C E 0 P 0 2 exp - 2 α e h / d s 2 P ,
F F e ϕ h / d s F = C E 0 P 0 2 exp - 2 α e h / d s 2 P ϕ h / d s F ,
ϕ h / d s F = a 4 h / d s F 4 + a 3 h / d s F 3 + a 2 h / d s F 2 + a 1 h / d s F + a 0
P 0 h / d s = P 0 exp α e h / d s 2 P ϕ - 1 / 2 h / d s F .
F     C E 0 P 0 2 exp - 2 α e h / d s 2 P exp - α f h / d s F = C E 0 P 0 2 exp - h 2 α e / d s 2 P + α f / d s f ,
P 0 h / d s     P 0 exp α e h / d s 2 P exp α f h / 2 d s F .
μ λ e = 750   nm = μ 2 P = 6.4   cm - 1 = 1 / d s 2 P , μ λ f = 465 nm = μ f = 30.9   cm - 1 = 1 / d s f ,
F     C E 0 P 0 2 exp - h 2 α e / d s 2 P + α f / d s f ,

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