Abstract

We present experiments to predict the maximum penetration depth at which typical biological structures in amelanotic tissue can be detected with confocal microscopy. The detected signal is examined as the signal source strength (index of refraction mismatch), the source depth, and the medium scattering coefficient are varied. The detected background produced by scattering outside the focal volume is examined as the medium scattering coefficient, the depth in the medium, the dimensionless pinhole radius, ν p, and the shape of the scattering phase function are varied. When the system approaches ideal confocal performance (ν p ≃ 3), the penetration depth is limited by the signal-to-noise ratio to approximately 3–4 optical depths (OD’s) for a 0.05 index mismatch. As ν p increases to 8, the penetration depth is limited by the signal-to-background ratio and is dependent on the scattering coefficient. At μs= 100 cm-1 (l s = 100 μm) and an index mismatch of 0.05, the maximum penetration depth is approximately 2 OD.

© 1998 Optical Society of America

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References

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1997 (1)

W. Starr, “Light dosimetry in vivo,” Phys. Med. Biol. 42, 763–787 (1997).
[CrossRef]

1996 (4)

1995 (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, 946–952 (1995).
[CrossRef] [PubMed]

1994 (5)

1993 (1)

1991 (1)

J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lamp, H. D. Cavanagh, “In vivo, real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
[CrossRef] [PubMed]

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, 946–952 (1995).
[CrossRef] [PubMed]

Andrews, P. M.

J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lamp, H. D. Cavanagh, “In vivo, real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
[CrossRef] [PubMed]

Bigio, I.

Boyer, J.

Brain, K.

Cavanagh, H. D.

J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lamp, H. D. Cavanagh, “In vivo, real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
[CrossRef] [PubMed]

Dunn, A. K.

A. K. Dunn, C. Smithpeter, A. Welch, R. Richards-Kortum, “Sources of contrast in confocal reflectance imaging,” Appl. Opt. 35, 3441–3446 (1996).
[CrossRef] [PubMed]

A. K. Dunn, R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1996).
[CrossRef]

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, 946–952 (1995).
[CrossRef] [PubMed]

Fantini, S.

Franceschini, M.

Gemert, M. V.

Gratton, E.

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, 946–952 (1995).
[CrossRef] [PubMed]

Gu, M.

Hee, M.

Hielscher, A.

Izatt, J.

Jester, J. V.

J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lamp, H. D. Cavanagh, “In vivo, real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
[CrossRef] [PubMed]

Kempe, M.

Knuttel, A.

Lamp, M. A.

J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lamp, H. D. Cavanagh, “In vivo, real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
[CrossRef] [PubMed]

Maier, J.

Masters, B.

Mourant, J.

Owen, G.

Petroll, W. M.

J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lamp, H. D. Cavanagh, “In vivo, real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
[CrossRef] [PubMed]

Prahl, S.

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, 946–952 (1995).
[CrossRef] [PubMed]

Richards-Kortum, R.

A. K. Dunn, C. Smithpeter, A. Welch, R. Richards-Kortum, “Sources of contrast in confocal reflectance imaging,” Appl. Opt. 35, 3441–3446 (1996).
[CrossRef] [PubMed]

A. K. Dunn, R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1996).
[CrossRef]

Rudolph, W.

Schmitt, J.

Sheppard, C. J.

Smithpeter, C.

Starr, W.

W. Starr, “Light dosimetry in vivo,” Phys. Med. Biol. 42, 763–787 (1997).
[CrossRef]

Thaer, A. A.

van de Hulst, H.

H. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1957).

Walker, S.

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, 946–952 (1995).
[CrossRef] [PubMed]

Welch, A.

Welsch, E.

Wilson, T.

T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 99–113.

Yadlowsky, M.

Zhou, H.

Appl. Opt. (4)

IEEE J. Sel. Top. Quantum Electron. (1)

A. K. Dunn, R. Richards-Kortum, “Three-dimensional computation of light scattering from cells,” IEEE J. Sel. Top. Quantum Electron. 2, 898–905 (1996).
[CrossRef]

J. Electron Microsc. Tech. (1)

J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lamp, H. D. Cavanagh, “In vivo, real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
[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, 946–952 (1995).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (2)

Opt. Lett. (3)

Phys. Med. Biol. (1)

W. Starr, “Light dosimetry in vivo,” Phys. Med. Biol. 42, 763–787 (1997).
[CrossRef]

Other (2)

H. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1957).

T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 99–113.

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Figures (8)

Fig. 1
Fig. 1

Experimental setup used to measure the confocal reflected light from tissue phantoms as a function of focal depth within the sample.

Fig. 2
Fig. 2

Tissue phantoms used to measure (a) the background from a uniform scattering layer of gelatin and (b) the signal and the background from uniform layers of gelatin (n = 1.36) and immersion oil (n = 1.41 and 1.45) with equivalent scattering coefficients.

Fig. 3
Fig. 3

Processed background signal measured with a ν p of 8 versus an optical depth for μ s of 46, 92, and 138 cm-1. The fitted exponential equations (lines) to the data (symbols) appear in the legend.

Fig. 4
Fig. 4

(a) Dependence of fitted B 0 on the scattering coefficient of the uniform phantom for ν p of 3 and 8. The fitted linear equations (lines) to the data (symbols) appear in the legend. (b) Dependence of fitted B 0 on ν p for a scattering coefficient of 100 cm-1.

Fig. 5
Fig. 5

(a) Dependence of fitted A B on the scattering coefficient of the uniform phantom for ν p of 3 and 8. The fitted linear equations (lines) to the data (symbols) appear in the legend. (b) Dependence of fitted A B on ν p for a scattering coefficient of 100 cm-1.

Fig. 6
Fig. 6

(a) Processed scan measured with a ν p of 8 from a multilayer phantom with a mismatch of 0.09 and a scattering coefficient of 138 cm-1. (b) Processed signal peak amplitude measured with a ν p of 8 versus optical depth for index mismatches of 0.05 and 0.09. Fitted exponential equations (lines) to the data (symbols) appear in the legend.

Fig. 7
Fig. 7

Maximum penetration depth limits due to S/B ratio limits calculated from Eq. (4) for (a) ν p = 8, (b) ν p = 3, and S/B ratio detection limits of 1, 2, and 4. Horizontal lines represent the penetration depth limits due to S/N ratio limits calculated from Eq. (5) for a S/N ratio detection limit of 2 and detector bandwidths of 63 kHz and 10 MHz.

Fig. 8
Fig. 8

Comparison of measured background from a tissue phantom (μ s = 92 cm-1) with the background predicted from a Monte Carlo model with two different phase functions with an anisotropy, g, of 0.9.

Equations (5)

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

B OD ,   μ s ,   ν p = B 0 μ s ,   ν p exp - A B μ s ,   ν p OD .
S OD = S 0 exp - A S OD ,
S OD B OD ,   μ s ,   ν p = S 0 exp - 2 OD B 0 μ s ,   ν p exp - A B μ s ,   ν p OD = S 0 B 0 μ s ,   ν p exp - 2 - A B μ s ,   ν p OD ,
OD Max , S / B = - ln S / B lim B 0 μ s ,   ν p + ln S 0 2 - A B μ s ,   ν p .
OD max , S / N = - ln S / N lim NEP G BW 1 / 2 + ln V 0 2 .

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