Abstract

We describe a new, nonapplanating, real-time scanning slit confocal microscope that produces unique real-time video images of the in vivo human cornea. This new real-time slit scanning confocal microscope produces en face, single-video-frame images (2-μm sections) with high contrast through the full thickness (500 μm) of the normal cornea. No frame averaging or digital image enhancement is required. The images of superficial epithelial cells, wing cells, basal epithelial cells, corneal innervation, nuclei of stromal keratocytes, and the cell bodies of the stromal keratocytes in the posterior stromal region demonstrate the unique real-time imaging characteristics. The microscope is equipped with water-immersion microscope objectives of high numerical aperture. The microscope objective does not flatten or distort the cornea; a polymer gel is used to couple the microscope objective optically to the cornea.

© 1994 Optical Society of America

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    [CrossRef]
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    [CrossRef]
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  12. G. S. Kino, G. Q. Xiao, “Real-time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.
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  17. B. R. Masters, “Two and three-dimensional visualization of the living cornea and ocular lens,” Mach. Vision Appl. 4, 227–232 (1991).
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  18. B. R. Masters, “Confocal microscopy of the in situ crystalline lens,” J. Microsc. 165, 159–167 (1992).
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  19. B. R. Masters, A. Kriete, J. Kukulies, “Ultraviolet confocal fluorescence microscopy of the in vitro cornea: redox metabolic imaging,” Appl. Opt. 32, 592–596 (1993).
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  20. B. R. Masters, M. A. Farmer, “Three-dimensional confocal microscopy and visualization of the in situ cornea,” Comput. Med. Imaging Graphics 17, 211–219 (1993).
    [CrossRef]
  21. G. Q. Xiao, G. S. Kino, B. R. Masters, “Observation of the rabbit cornea and lens with a new real-time confocal scanning optical microscope,” Scanning 12, 161–166 (1990).
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  22. J. V. Jester, H. D. Cavanagh, M. A. Lemp, “In vivo confocal imaging of the eye using tandem scanning confocal microscopy (TSCM),” in Scanning Imaging, Tony Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 122–126 (1988).
  23. J. V. Jester, H. D. Cavanagh, J. Essepian, W. J. Shields, M. A. Lemp, “Real-time confocal imaging of the living eye,” in New Methods in Microscopy and Low Light Imaging, J. E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 366–376 (1989).
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  25. R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).
  26. A. R. Wegener, G. Gaida, J. H. Massig, A. Breipohl, “In-vivo microscopical investigation of cornea and lens by confocal optics,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).
  27. C. J. Koester, C. W. Roberts, “Wide-field specular microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 99–121.
    [CrossRef]
  28. C. J. Koester, S. M. Khanna, “Optical sectioning with the scanning slit confocal microscope: applications in ophthalmology and ear research,” in New Methods in Microscopy and Low Light Imaging, John E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 378–389 (1989).
  29. G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).
  30. C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).
  31. C. J. Koester, “Confocal microscopy of the cornea,” in Ophthalmic and Visual Optics and Noninvasive Assessment of the Visual System, Vol. 3 of 1993 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1993), p. 132.
  32. B. R. Masters, A. A. Thaer, “Confocal microscopy of the human in vivo cornea,” in Ophthalmic and Visual Optics and Noninvasive Assessment of the Visual System, Vol. 3 of 1993 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1993), pp. 133–136 (1993).
  33. A. A. Thaer, O.-C. Geyer, F. A. Kaszli, “Fluorogenic substrate techniques applied to the noninvasive diagnosis of the living rabbit and human cornea,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 569–590.
    [CrossRef]
  34. B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the human cornea,” Biophys. J. 64, A108 (1993).
  35. B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the living human eye: a new real-time confocal system,” presented at the 1993 International Conference on Confocal Microscopy and Three-Dimensional Image Processing, Sydney, Australia, 1993.
  36. G. M. Svishchev, “Microscope for the study of transparent light-scattering objects in incident light,” Opt. Spectrosc. 26, 171–172 (1969).
  37. G. J. Brakenhoff, K. Visscher, “Confocal imaging with bilateral scanning and array detectors,” J. Microsc. 165, 139–146(1992).
    [CrossRef]
  38. J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lemp, D. Cavanagh, “In vivo real-time confocal imaging,” J. Electron Microsc. Tech. 18, 50–60 (1991).
    [CrossRef] [PubMed]
  39. J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
    [CrossRef] [PubMed]

1993 (3)

B. R. Masters, A. Kriete, J. Kukulies, “Ultraviolet confocal fluorescence microscopy of the in vitro cornea: redox metabolic imaging,” Appl. Opt. 32, 592–596 (1993).
[CrossRef] [PubMed]

B. R. Masters, M. A. Farmer, “Three-dimensional confocal microscopy and visualization of the in situ cornea,” Comput. Med. Imaging Graphics 17, 211–219 (1993).
[CrossRef]

B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the human cornea,” Biophys. J. 64, A108 (1993).

1992 (7)

G. J. Brakenhoff, K. Visscher, “Confocal imaging with bilateral scanning and array detectors,” J. Microsc. 165, 139–146(1992).
[CrossRef]

J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
[CrossRef] [PubMed]

B. R. Masters, “Confocal microscopy of the in situ crystalline lens,” J. Microsc. 165, 159–167 (1992).
[CrossRef] [PubMed]

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

A. R. Wegener, G. Gaida, J. H. Massig, A. Breipohl, “In-vivo microscopical investigation of cornea and lens by confocal optics,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).

C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).

1991 (2)

B. R. Masters, “Two and three-dimensional visualization of the living cornea and ocular lens,” Mach. Vision Appl. 4, 227–232 (1991).
[CrossRef]

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

1990 (3)

G. Q. Xiao, G. S. Kino, B. R. Masters, “Observation of the rabbit cornea and lens with a new real-time confocal scanning optical microscope,” Scanning 12, 161–166 (1990).
[CrossRef]

B. R. Masters, S. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–274 (1990).
[CrossRef] [PubMed]

B. R. Masters, S. W. Paddock, “Three-dimensional reconstruction of the rabbit cornea by confocal scanning optical microscopy and volume rendering,” Appl. Opt. 29, 3816–3822 (1990).
[CrossRef] [PubMed]

1988 (1)

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

1986 (1)

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscopy of the full-thickness cornea,” Cornea 4, 205–209(1986).

1985 (1)

M. Petran, M. Hadravsky, A. Boyde, “The tandem scanning reflected light microscope,” Scanning 7, 97–108 (1985).
[CrossRef]

1980 (1)

1974 (1)

D. M. Maurice, “A scanning slit optical microscope,” Invest. Ophthalmol. 13, 1033–1037 (1974).
[PubMed]

1969 (1)

G. M. Svishchev, “Microscope for the study of transparent light-scattering objects in incident light,” Opt. Spectrosc. 26, 171–172 (1969).

1968 (1)

Andrews, P. M.

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

Assouline, M.

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Auran, J. D.

G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).

C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).

Baer, S. C.

S. C. Baer, “Optical apparatus providing focal-plane-specific illumination,” U.S. patent3,547,512 (15December1970).

Barron, B.

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Beuerman, R. W.

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Boyde, A.

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscopy of the full-thickness cornea,” Cornea 4, 205–209(1986).

M. Petran, M. Hadravsky, A. Boyde, “The tandem scanning reflected light microscope,” Scanning 7, 97–108 (1985).
[CrossRef]

Brady, J.

G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).

Brakenhoff, G. J.

G. J. Brakenhoff, K. Visscher, “Confocal imaging with bilateral scanning and array detectors,” J. Microsc. 165, 139–146(1992).
[CrossRef]

Breipohl, A.

A. R. Wegener, G. Gaida, J. H. Massig, A. Breipohl, “In-vivo microscopical investigation of cornea and lens by confocal optics,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Cavanagh, D.

J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
[CrossRef] [PubMed]

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

Cavanagh, H. D.

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “Confocal microscopic imaging of the living eye with tandem scanning confocal microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 172–188.
[CrossRef]

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “In vivo confocal imaging of the eye using tandem scanning confocal microscopy (TSCM),” in Scanning Imaging, Tony Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 122–126 (1988).

J. V. Jester, H. D. Cavanagh, J. Essepian, W. J. Shields, M. A. Lemp, “Real-time confocal imaging of the living eye,” in New Methods in Microscopy and Low Light Imaging, J. E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 366–376 (1989).

Chew, S. J.

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Corle, T. R.

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

Dilly, P. N.

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscopy of the full-thickness cornea,” Cornea 4, 205–209(1986).

Egger, M. D.

Essepian, J.

J. V. Jester, H. D. Cavanagh, J. Essepian, W. J. Shields, M. A. Lemp, “Real-time confocal imaging of the living eye,” in New Methods in Microscopy and Low Light Imaging, J. E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 366–376 (1989).

Farmer, M. A.

B. R. Masters, M. A. Farmer, “Three-dimensional confocal microscopy and visualization of the in situ cornea,” Comput. Med. Imaging Graphics 17, 211–219 (1993).
[CrossRef]

Florakis, G. J.

G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).

Gaida, G.

A. R. Wegener, G. Gaida, J. H. Massig, A. Breipohl, “In-vivo microscopical investigation of cornea and lens by confocal optics,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Galambos, R.

Garana, R. M. R.

J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
[CrossRef] [PubMed]

Geyer, O.-C.

A. A. Thaer, O.-C. Geyer, F. A. Kaszli, “Fluorogenic substrate techniques applied to the noninvasive diagnosis of the living rabbit and human cornea,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 569–590.
[CrossRef]

Gullstrand, A.

A. Gullstrand, Demonstration der Nerstspalt (Heidelbergen Bericht, Heidelberg, 1911).

Hadravsky, M.

M. Petran, M. Hadravsky, A. Boyde, “The tandem scanning reflected light microscope,” Scanning 7, 97–108 (1985).
[CrossRef]

M. Petran, M. Hadravsky, M. D. Egger, R. Galambos, “Tandem-scanning reflected-light microscopy,” J. Opt. Soc. Am. 58, 661–664 (1968).
[CrossRef]

Hill, J.

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Jester, J. V.

J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
[CrossRef] [PubMed]

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

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “Confocal microscopic imaging of the living eye with tandem scanning confocal microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 172–188.
[CrossRef]

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “In vivo confocal imaging of the eye using tandem scanning confocal microscopy (TSCM),” in Scanning Imaging, Tony Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 122–126 (1988).

J. V. Jester, H. D. Cavanagh, J. Essepian, W. J. Shields, M. A. Lemp, “Real-time confocal imaging of the living eye,” in New Methods in Microscopy and Low Light Imaging, J. E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 366–376 (1989).

Kaszli, F. A.

A. A. Thaer, O.-C. Geyer, F. A. Kaszli, “Fluorogenic substrate techniques applied to the noninvasive diagnosis of the living rabbit and human cornea,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 569–590.
[CrossRef]

Kaufman, H. E.

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Khanna, S. M.

C. J. Koester, S. M. Khanna, “Optical sectioning with the scanning slit confocal microscope: applications in ophthalmology and ear research,” in New Methods in Microscopy and Low Light Imaging, John E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 378–389 (1989).

Kino, G. S.

G. Q. Xiao, G. S. Kino, B. R. Masters, “Observation of the rabbit cornea and lens with a new real-time confocal scanning optical microscope,” Scanning 12, 161–166 (1990).
[CrossRef]

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

G. S. Kino, G. Q. Xiao, “Real-time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.

Koester, C. J.

C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).

G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).

C. J. Koester, “Scanning mirror microscope with optical sectioning characteristics: applications to ophthalmology,” Appl. Opt. 19, 1749–1757 (1980).
[CrossRef] [PubMed]

C. J. Koester, S. M. Khanna, “Optical sectioning with the scanning slit confocal microscope: applications in ophthalmology and ear research,” in New Methods in Microscopy and Low Light Imaging, John E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 378–389 (1989).

C. J. Koester, C. W. Roberts, “Wide-field specular microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 99–121.
[CrossRef]

C. J. Koester, “Confocal microscopy of the cornea,” in Ophthalmic and Visual Optics and Noninvasive Assessment of the Visual System, Vol. 3 of 1993 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1993), p. 132.

Kriete, A.

Kukulies, J.

Lemp, M. A.

J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
[CrossRef] [PubMed]

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

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscopy of the full-thickness cornea,” Cornea 4, 205–209(1986).

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “In vivo confocal imaging of the eye using tandem scanning confocal microscopy (TSCM),” in Scanning Imaging, Tony Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 122–126 (1988).

J. V. Jester, H. D. Cavanagh, J. Essepian, W. J. Shields, M. A. Lemp, “Real-time confocal imaging of the living eye,” in New Methods in Microscopy and Low Light Imaging, J. E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 366–376 (1989).

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “Confocal microscopic imaging of the living eye with tandem scanning confocal microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 172–188.
[CrossRef]

Massig, J. H.

A. R. Wegener, G. Gaida, J. H. Massig, A. Breipohl, “In-vivo microscopical investigation of cornea and lens by confocal optics,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Masters, B. R.

B. R. Masters, M. A. Farmer, “Three-dimensional confocal microscopy and visualization of the in situ cornea,” Comput. Med. Imaging Graphics 17, 211–219 (1993).
[CrossRef]

B. R. Masters, A. Kriete, J. Kukulies, “Ultraviolet confocal fluorescence microscopy of the in vitro cornea: redox metabolic imaging,” Appl. Opt. 32, 592–596 (1993).
[CrossRef] [PubMed]

B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the human cornea,” Biophys. J. 64, A108 (1993).

B. R. Masters, “Confocal microscopy of the in situ crystalline lens,” J. Microsc. 165, 159–167 (1992).
[CrossRef] [PubMed]

B. R. Masters, “Two and three-dimensional visualization of the living cornea and ocular lens,” Mach. Vision Appl. 4, 227–232 (1991).
[CrossRef]

B. R. Masters, S. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–274 (1990).
[CrossRef] [PubMed]

B. R. Masters, S. W. Paddock, “Three-dimensional reconstruction of the rabbit cornea by confocal scanning optical microscopy and volume rendering,” Appl. Opt. 29, 3816–3822 (1990).
[CrossRef] [PubMed]

G. Q. Xiao, G. S. Kino, B. R. Masters, “Observation of the rabbit cornea and lens with a new real-time confocal scanning optical microscope,” Scanning 12, 161–166 (1990).
[CrossRef]

B. R. Masters, “Effects of contact lenses on the oxygen concentration and epithelial mitochondrial redox state of rabbit cornea measured noninvasively with and optically sectioning redox fluorometer microscope,” in The Cornea: Transactions of the World Congress on the Cornea III, H. D. Cavanagh, ed. (Raven, New York, 1988), pp. 281–286.

B. R. Masters, A. A. Thaer, “Confocal microscopy of the human in vivo cornea,” in Ophthalmic and Visual Optics and Noninvasive Assessment of the Visual System, Vol. 3 of 1993 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1993), pp. 133–136 (1993).

B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the living human eye: a new real-time confocal system,” presented at the 1993 International Conference on Confocal Microscopy and Three-Dimensional Image Processing, Sydney, Australia, 1993.

Maurice, D. M.

D. M. Maurice, “A scanning slit optical microscope,” Invest. Ophthalmol. 13, 1033–1037 (1974).
[PubMed]

Minsky, M.

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (19December1961).

Nipkow, P.

P. Nipkow, “Optical disk,” German patent30,105 (15January1884).

Norris, W. F.

W. F. Norris, C. A. Oliver, System of Diseases of the Eye (Lippincott, Philadelphia, Pa., 1897).

Oliver, C. A.

W. F. Norris, C. A. Oliver, System of Diseases of the Eye (Lippincott, Philadelphia, Pa., 1897).

Paddock, S.

B. R. Masters, S. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–274 (1990).
[CrossRef] [PubMed]

Paddock, S. W.

Pedroza, L.

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Petran, M.

M. Petran, M. Hadravsky, A. Boyde, “The tandem scanning reflected light microscope,” Scanning 7, 97–108 (1985).
[CrossRef]

M. Petran, M. Hadravsky, M. D. Egger, R. Galambos, “Tandem-scanning reflected-light microscopy,” J. Opt. Soc. Am. 58, 661–664 (1968).
[CrossRef]

Petroll, V. M.

J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
[CrossRef] [PubMed]

Petroll, W. M.

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

Roberts, C. W.

C. J. Koester, C. W. Roberts, “Wide-field specular microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 99–121.
[CrossRef]

Rosskothen, H. D.

C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).

Shields, W. J.

J. V. Jester, H. D. Cavanagh, J. Essepian, W. J. Shields, M. A. Lemp, “Real-time confocal imaging of the living eye,” in New Methods in Microscopy and Low Light Imaging, J. E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 366–376 (1989).

Svishchev, G. M.

G. M. Svishchev, “Microscope for the study of transparent light-scattering objects in incident light,” Opt. Spectrosc. 26, 171–172 (1969).

Tackaberry, R. B.

C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).

Thaer, A. A.

B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the human cornea,” Biophys. J. 64, A108 (1993).

B. R. Masters, A. A. Thaer, “Confocal microscopy of the human in vivo cornea,” in Ophthalmic and Visual Optics and Noninvasive Assessment of the Visual System, Vol. 3 of 1993 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1993), pp. 133–136 (1993).

A. A. Thaer, O.-C. Geyer, F. A. Kaszli, “Fluorogenic substrate techniques applied to the noninvasive diagnosis of the living rabbit and human cornea,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 569–590.
[CrossRef]

B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the living human eye: a new real-time confocal system,” presented at the 1993 International Conference on Confocal Microscopy and Three-Dimensional Image Processing, Sydney, Australia, 1993.

Trokel, S. L.

C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).

G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).

Visscher, K.

G. J. Brakenhoff, K. Visscher, “Confocal imaging with bilateral scanning and array detectors,” J. Microsc. 165, 139–146(1992).
[CrossRef]

Vogt, A.

A. Vogt, Lehrbuch und Atlas der Spaltlampen Mikroskopie des Lebenden Auges (Springer-Verlag, Berlin, 1930), Vol. 1, pp. 1–489.
[CrossRef]

Wegener, A. R.

A. R. Wegener, G. Gaida, J. H. Massig, A. Breipohl, “In-vivo microscopical investigation of cornea and lens by confocal optics,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Xiao, G. Q.

G. Q. Xiao, G. S. Kino, B. R. Masters, “Observation of the rabbit cornea and lens with a new real-time confocal scanning optical microscope,” Scanning 12, 161–166 (1990).
[CrossRef]

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

G. S. Kino, G. Q. Xiao, “Real-time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.

Appl. Opt. (3)

Appl. Phys. Lett. (1)

G. Q. Xiao, T. R. Corle, G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[CrossRef]

Biophys. J. (1)

B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the human cornea,” Biophys. J. 64, A108 (1993).

Comput. Med. Imaging Graphics (1)

B. R. Masters, M. A. Farmer, “Three-dimensional confocal microscopy and visualization of the in situ cornea,” Comput. Med. Imaging Graphics 17, 211–219 (1993).
[CrossRef]

Cornea (1)

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscopy of the full-thickness cornea,” Cornea 4, 205–209(1986).

Invest. Ophthalmol. (1)

D. M. Maurice, “A scanning slit optical microscope,” Invest. Ophthalmol. 13, 1033–1037 (1974).
[PubMed]

Invest. Ophthalmol. Visual Sci. (1)

A. R. Wegener, G. Gaida, J. H. Massig, A. Breipohl, “In-vivo microscopical investigation of cornea and lens by confocal optics,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

Invest. Ophthalmol. Visual Sci. (3)

R. W. Beuerman, S. J. Chew, L. Pedroza, M. Assouline, B. Barron, J. Hill, H. E. Kaufman, “Early diagnosis of infectious keratitis with in vivo real-time confocal microscopy,” Invest. Ophthalmol. Visual Sci. 33, 1234 (1992).

G. J. Florakis, J. D. Auran, C. J. Koester, J. Brady, S. L. Trokel, “High resolution scanning slit microscopy of the human cornea epithelium in vivo,” Invest. Ophthalmol. Visual Sci. 33, 1234(1992).

C. J. Koester, J. D. Auran, H. D. Rosskothen, S. L. Trokel, R. B. Tackaberry, “A contact microscope objective with high numerical aperture for clinical examination of the cornea,” Invest. Ophthalmol. Visual Sci. 33, 1233 (1992).

J. Electron Microsc. Tech. (1)

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

J. Microsc. (4)

J. V. Jester, V. M. Petroll, R. M. R. Garana, M. A. Lemp, D. Cavanagh, “Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. 165, 169–181 (1992).
[CrossRef] [PubMed]

G. J. Brakenhoff, K. Visscher, “Confocal imaging with bilateral scanning and array detectors,” J. Microsc. 165, 139–146(1992).
[CrossRef]

B. R. Masters, “Confocal microscopy of the in situ crystalline lens,” J. Microsc. 165, 159–167 (1992).
[CrossRef] [PubMed]

B. R. Masters, S. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–274 (1990).
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

Mach. Vision Appl. (1)

B. R. Masters, “Two and three-dimensional visualization of the living cornea and ocular lens,” Mach. Vision Appl. 4, 227–232 (1991).
[CrossRef]

Opt. Spectrosc. (1)

G. M. Svishchev, “Microscope for the study of transparent light-scattering objects in incident light,” Opt. Spectrosc. 26, 171–172 (1969).

Scanning (2)

G. Q. Xiao, G. S. Kino, B. R. Masters, “Observation of the rabbit cornea and lens with a new real-time confocal scanning optical microscope,” Scanning 12, 161–166 (1990).
[CrossRef]

M. Petran, M. Hadravsky, A. Boyde, “The tandem scanning reflected light microscope,” Scanning 7, 97–108 (1985).
[CrossRef]

Other (17)

G. S. Kino, G. Q. Xiao, “Real-time scanning optical microscopes,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 361–387.

S. C. Baer, “Optical apparatus providing focal-plane-specific illumination,” U.S. patent3,547,512 (15December1970).

B. R. Masters, “Effects of contact lenses on the oxygen concentration and epithelial mitochondrial redox state of rabbit cornea measured noninvasively with and optically sectioning redox fluorometer microscope,” in The Cornea: Transactions of the World Congress on the Cornea III, H. D. Cavanagh, ed. (Raven, New York, 1988), pp. 281–286.

A. Vogt, Lehrbuch und Atlas der Spaltlampen Mikroskopie des Lebenden Auges (Springer-Verlag, Berlin, 1930), Vol. 1, pp. 1–489.
[CrossRef]

W. F. Norris, C. A. Oliver, System of Diseases of the Eye (Lippincott, Philadelphia, Pa., 1897).

A. Gullstrand, Demonstration der Nerstspalt (Heidelbergen Bericht, Heidelberg, 1911).

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (19December1961).

P. Nipkow, “Optical disk,” German patent30,105 (15January1884).

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “In vivo confocal imaging of the eye using tandem scanning confocal microscopy (TSCM),” in Scanning Imaging, Tony Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 122–126 (1988).

J. V. Jester, H. D. Cavanagh, J. Essepian, W. J. Shields, M. A. Lemp, “Real-time confocal imaging of the living eye,” in New Methods in Microscopy and Low Light Imaging, J. E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 366–376 (1989).

J. V. Jester, H. D. Cavanagh, M. A. Lemp, “Confocal microscopic imaging of the living eye with tandem scanning confocal microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 172–188.
[CrossRef]

C. J. Koester, C. W. Roberts, “Wide-field specular microscopy,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 99–121.
[CrossRef]

C. J. Koester, S. M. Khanna, “Optical sectioning with the scanning slit confocal microscope: applications in ophthalmology and ear research,” in New Methods in Microscopy and Low Light Imaging, John E. Wampler, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1161, 378–389 (1989).

B. R. Masters, A. A. Thaer, “In vivo confocal microscopy of the living human eye: a new real-time confocal system,” presented at the 1993 International Conference on Confocal Microscopy and Three-Dimensional Image Processing, Sydney, Australia, 1993.

C. J. Koester, “Confocal microscopy of the cornea,” in Ophthalmic and Visual Optics and Noninvasive Assessment of the Visual System, Vol. 3 of 1993 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1993), p. 132.

B. R. Masters, A. A. Thaer, “Confocal microscopy of the human in vivo cornea,” in Ophthalmic and Visual Optics and Noninvasive Assessment of the Visual System, Vol. 3 of 1993 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1993), pp. 133–136 (1993).

A. A. Thaer, O.-C. Geyer, F. A. Kaszli, “Fluorogenic substrate techniques applied to the noninvasive diagnosis of the living rabbit and human cornea,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 569–590.
[CrossRef]

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Figures (11)

Fig. 1
Fig. 1

Optical system of the real-time scanning slit confocal microscope. The light source is a halogen lamp. For fluorescence confocal microscopy, a 100-W mercury arc lamp can replace the halogen lamp. Fl, infrared filter; S1, adjustable iris; S2, S3, confocal slits; L’s, lenses. Objective shows the position of the microscope objective. M1–M6, fixed front surface mirrors; M7, oscillating double-sided mirror that is used for both scanning and descanning. Camera is the position of the intensified video camera. The video monitor and the video recorder are not shown.

Fig. 2
Fig. 2

In vivo confocal microscopic en face image of a human cornea. The U-matic tape was stopped on a single video frame by the pause mode, and a 35-mm film camera was used to photograph the television monitor to produce the image. No digital or analog image enhancement or processing was used. There was no frame averaging applied to the video images. The photograph shows the raw data photographed directly from the screen of the television monitor. All the figures were made under the same conditions (see section on Methods). This figure shows a single video frame of the superficial cells of the corneal epithelium. This cell layer, which is just below the tear film, has the following characteristics: The cell nucleus appears as a highly reflecting bright oval that is surrounded by a darker band. The focal plane is set to section optically the most superficial cells that appear brightest; cells slightly below this focal plane appear darker. 25× objective, 0.6 NA.

Fig. 3
Fig. 3

In vivo confocal microscopic en face image of a human cornea. The optical section is of a plane of cells just below that shown in Fig. 2. The bright oval bodies are cell nuclei (see arrowheads) of the superficial cells. 25× objective, 0.6 NA.

Fig. 4
Fig. 4

In vivo confocal microscopic en face image of a human cornea. This optical section is in the plane of the wing cells that are located between the superficial epithelial cells and the basal epithelial cells of the human cornea. The image of these cells is characterized by the bright cell nuclei (see arrowhead) that are devoid of the darker band that is observed in the superficial epithelial cells. 50× objective, 1.0 NA.

Fig. 5
Fig. 5

In vivo confocal microscopic en face image of a human cornea. The optical section is in the plane of the basal epithelial cells. The image shows the bright cell borders and the darker cell interiors. The cell borders appear thick because there is a high degree of invagination between the adjacent cells. 25× water-immersion microscope objective, 0.6 NA.

Fig. 6
Fig. 6

In vivo confocal microscopic en face image of a human cornea. This is an optical section of the basal epithelial cells similar to that shown in Fig. 5. The higher magnification is obtained with the use of a water-immersion microscope objective of power 50×, 1.0 NA. The higher NA of the microscope objective results in a thinner optical section with higher contrast than that in Fig. 5. Cell nuclei within basal epithelial cells are shown (see arrowheads). (This figure is reproduced at 100%. Figures 2 5 and 7 11 are reproduced at 69%.)

Fig. 7
Fig. 7

In vivo confocal microscopic en face image of a human cornea. This optical section shows the corneal nerves in the anterior stroma just below Bowman’s membrane. The bright objects are the nuclei of the stromal keratocytes. The varicosities of the stromal nerve trunk are shown along the length of the nerve (see arrowheads). 50× water-immersion microscope objective, 1.0 NA.

Fig. 8
Fig. 8

In vivo confocal microscopic en face image of a human cornea. This optical section shows the nuclei of the stromal keratocytes at the midstromal region. The optical section is set to be minimal by closing down the slits. Two bright nuclei of stromal keratocytes are shown. Each nucleus has a dark line in the center (see arrow) that is a nuclear cleft in which the cytoplasm forms a cleft on the surface of the nucleus. The cleft is shown as a dark line. 50× water-immersion microscope objective, 1.0 NA.

Fig. 9
Fig. 9

In vivo confocal microscopic en face image of a normal human cornea. This optical section shows the nuclei of the stromal keratocytes in the posterior stroma near Descemet’s membrane (~500 μm below the surface of the cornea). The bright oval objects are the nuclei of the stromal keratocytes (see star). The cell bodies of the stromal keratocytes are also shown. Reflected light from the corneal endothelium back illuminates the stromal keratocytes and forms the image of nuclei and cell bodies. 50× objective, 1.0 NA.

Fig. 10
Fig. 10

In vivo confocal microscopic en face image of a normal human cornea. The optical section shows the nuclei (see star) of the stromal keratocyte in the posterior stroma near Descemet’s membrane. This figure illustrates the diverse shapes of keratocyte nuclei. 50× water-immersion microscope objective, 1.0 NA.

Fig. 11
Fig. 11

In vivo confocal microscopic en face image of a humai cornea. This optical section shows the polygonal corneal endothe liai cells. This image is 500 μm below the surface of the corne (Fig. 2). The three black regions represent endothelial defects The focal plane is between the posterior cornea and the aqueou humor. 50× water-immersion microscope objective, 1.0 NA.

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