Abstract

A laser scanning microscope was fitted with two argon-ion lasers that provided wavelengths in the regions of 364, 488, and 514 nm. A Zeiss water objective of 25 ×, with a numerical aperture of 0.8, corrected for the UV, was used to measure the fluorescence from optical sections of freshly enucleated rabbit eyes. The confocal microscope was used in both the reflected and fluorescent modes to image in situ epithelial and endothelial cells. An excitation wavelength of 364 nm and emission at 400–500 nm were used to image the fluorescence from reduced pyridine nucleotides. We demonstrate the feasibility of two-dimensional fluorescent confocal imaging of reduced pyridine nucleotides in corneal epithelial and endothelial cells.

© 1993 Optical Society of America

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References

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  1. B. Chance, B. Thorell, “Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry,” J. Biol. Chem. 234, 3044–3050 (1959).
    [PubMed]
  2. B. Chance, M. Lieberman, “Intrinsic fluorescence emission from the cornea at low temperatures: evidence of mitochondrial signals and their differing redox states in epithelial and endothelial sides,” Exp. Eye Res. 26, 111–117 (1978).
    [CrossRef] [PubMed]
  3. B. R. Masters, “Noninvasive redox fluorometry: how light can be used to monitor alterations of corneal mitochondrial function,” Curr. Eye Res. 3, 23–26 (1984).
    [CrossRef] [PubMed]
  4. B. R. Masters, “In vitro corneal redox fluorometry,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 223–247.
    [CrossRef]
  5. B. R. Masters, S. Falk, B. Chance, “In vivo flavoprotein redox measurements of rabbit corneal normoxic–anoxic transitions,” Curr. Eye Res. 1, 623–627 (1982).
    [CrossRef]
  6. B. R. Masters, “Noninvasive corneal redox fluorometry,” in Current Topics in Eye Research, J. Zadunaisky, H. Davson, eds. (Academic, London, 1984), pp. 139–200.
  7. B. R. Masters, “Confocal microscopy of ocular tissue,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990, pp. 305–324.
  8. B. R. Masters, “Effects of contact lenses on the oxygen concentration and epithelial mitochondrial redox state of rabbit cornea measured noninvasively with an 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. 2810–3860.
  9. B. R. Masters, M. V. Riley, J. Fischbarg, B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, noninvasive fluorometry and physiological correlates,” Exp. Eye Res. 36, 1–9 (1983).
    [CrossRef]
  10. B. R. Masters, “An optical method for the determination of oxygen concentration in the tear film,” in The Precorneal Tear Film in Health, Disease and Contact Lens Wear, F. Holly, ed. (Dry Eye Institute, Lubbock, Tex.1986), pp. 966–970.
  11. J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
    [CrossRef] [PubMed]
  12. T. Wilson, “Optical aspects of confocal microscopy,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 93–141.
  13. T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
    [CrossRef]
  14. S. Kimura, C. Munakata, “Depth resolution of the fluorescent confocal scanning optical microscope,” Appl. Opt. 29, 489–494 (1990).
    [CrossRef] [PubMed]
  15. S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc. Am. 6, 1015–1019 (1989).
    [CrossRef]
  16. S. D. Bennet, E. A. Peltzer, I. R. Smith, “Ultra-violet confocal metrology,” in Scanning Microscopy Technologies and Applications, E. C. Teague, ed., Proc. Soc. Photo-Opt. Instrum. Eng.897, 75–82 (1988).
  17. P. A. Heimann, R. Urstadt, “Deep ultraviolet microscope,” Appl. Opt. 29, 495–501 (1990).
    [CrossRef] [PubMed]
  18. M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscope for optically sectioning the living cornea,” Cornea 4, 205–209 (1986).
  19. B. R. Masters, S. W. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–275 (1990).
    [CrossRef] [PubMed]
  20. B. R. Masters, G. S. Kino, “Confocal microscopy of the eye,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 152–171.
    [CrossRef]
  21. B. R. Masters, G. S. Kino, “Real-time confocal scanning imaging of the eye: instrument performance of reflection and fluorescence imaging,” Proc. Inst. Phys. (UK) 98, 625–628 (1989).
  22. 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]
  23. A. Kriete, B. R. Masters, “Three-dimensional visualization of the living cornea,” in Transactions of the Royal Microscopical Society, MICRO 90, V. Howard, ed. (Institute of Physics, Bristol, UK, 1990), Vol. 1, pp. 401–404.
  24. 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]
  25. M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
    [CrossRef] [PubMed]
  26. H. G. Kapitza, V. Wilke, “Applications of the microscope system LSM,” in Scanning Imaging, T. Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 173–179 (1988).

1991

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

1990

1989

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc. Am. 6, 1015–1019 (1989).
[CrossRef]

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

B. R. Masters, G. S. Kino, “Real-time confocal scanning imaging of the eye: instrument performance of reflection and fluorescence imaging,” Proc. Inst. Phys. (UK) 98, 625–628 (1989).

1986

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscope for optically sectioning the living cornea,” Cornea 4, 205–209 (1986).

1984

B. R. Masters, “Noninvasive redox fluorometry: how light can be used to monitor alterations of corneal mitochondrial function,” Curr. Eye Res. 3, 23–26 (1984).
[CrossRef] [PubMed]

1983

B. R. Masters, M. V. Riley, J. Fischbarg, B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, noninvasive fluorometry and physiological correlates,” Exp. Eye Res. 36, 1–9 (1983).
[CrossRef]

1982

B. R. Masters, S. Falk, B. Chance, “In vivo flavoprotein redox measurements of rabbit corneal normoxic–anoxic transitions,” Curr. Eye Res. 1, 623–627 (1982).
[CrossRef]

1978

B. Chance, M. Lieberman, “Intrinsic fluorescence emission from the cornea at low temperatures: evidence of mitochondrial signals and their differing redox states in epithelial and endothelial sides,” Exp. Eye Res. 26, 111–117 (1978).
[CrossRef] [PubMed]

1959

B. Chance, B. Thorell, “Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry,” J. Biol. Chem. 234, 3044–3050 (1959).
[PubMed]

Balaban, R. S.

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

Bennet, S. D.

S. D. Bennet, E. A. Peltzer, I. R. Smith, “Ultra-violet confocal metrology,” in Scanning Microscopy Technologies and Applications, E. C. Teague, ed., Proc. Soc. Photo-Opt. Instrum. Eng.897, 75–82 (1988).

Boyde, A.

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscope for optically sectioning the living cornea,” Cornea 4, 205–209 (1986).

Chance, B.

B. R. Masters, M. V. Riley, J. Fischbarg, B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, noninvasive fluorometry and physiological correlates,” Exp. Eye Res. 36, 1–9 (1983).
[CrossRef]

B. R. Masters, S. Falk, B. Chance, “In vivo flavoprotein redox measurements of rabbit corneal normoxic–anoxic transitions,” Curr. Eye Res. 1, 623–627 (1982).
[CrossRef]

B. Chance, M. Lieberman, “Intrinsic fluorescence emission from the cornea at low temperatures: evidence of mitochondrial signals and their differing redox states in epithelial and endothelial sides,” Exp. Eye Res. 26, 111–117 (1978).
[CrossRef] [PubMed]

B. Chance, B. Thorell, “Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry,” J. Biol. Chem. 234, 3044–3050 (1959).
[PubMed]

Dilly, P. N.

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscope for optically sectioning the living cornea,” Cornea 4, 205–209 (1986).

Eng, J.

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

Falk, S.

B. R. Masters, S. Falk, B. Chance, “In vivo flavoprotein redox measurements of rabbit corneal normoxic–anoxic transitions,” Curr. Eye Res. 1, 623–627 (1982).
[CrossRef]

Fischbarg, J.

B. R. Masters, M. V. Riley, J. Fischbarg, B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, noninvasive fluorometry and physiological correlates,” Exp. Eye Res. 36, 1–9 (1983).
[CrossRef]

Gundlach, H.

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

Heimann, P. A.

Jorgens, R.

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

Kapitza, H. G.

H. G. Kapitza, V. Wilke, “Applications of the microscope system LSM,” in Scanning Imaging, T. Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 173–179 (1988).

Kimura, S.

S. Kimura, C. Munakata, “Depth resolution of the fluorescent confocal scanning optical microscope,” Appl. Opt. 29, 489–494 (1990).
[CrossRef] [PubMed]

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc. Am. 6, 1015–1019 (1989).
[CrossRef]

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]

B. R. Masters, G. S. Kino, “Real-time confocal scanning imaging of the eye: instrument performance of reflection and fluorescence imaging,” Proc. Inst. Phys. (UK) 98, 625–628 (1989).

B. R. Masters, G. S. Kino, “Confocal microscopy of the eye,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 152–171.
[CrossRef]

Kriete, A.

A. Kriete, B. R. Masters, “Three-dimensional visualization of the living cornea,” in Transactions of the Royal Microscopical Society, MICRO 90, V. Howard, ed. (Institute of Physics, Bristol, UK, 1990), Vol. 1, pp. 401–404.

Kukulies, J.

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

Lemp, M. A.

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscope for optically sectioning the living cornea,” Cornea 4, 205–209 (1986).

Lieberman, M.

B. Chance, M. Lieberman, “Intrinsic fluorescence emission from the cornea at low temperatures: evidence of mitochondrial signals and their differing redox states in epithelial and endothelial sides,” Exp. Eye Res. 26, 111–117 (1978).
[CrossRef] [PubMed]

Lynch, R. M.

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

Masters, B. R.

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]

B. R. Masters, S. W. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–275 (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, G. S. Kino, “Real-time confocal scanning imaging of the eye: instrument performance of reflection and fluorescence imaging,” Proc. Inst. Phys. (UK) 98, 625–628 (1989).

B. R. Masters, “Noninvasive redox fluorometry: how light can be used to monitor alterations of corneal mitochondrial function,” Curr. Eye Res. 3, 23–26 (1984).
[CrossRef] [PubMed]

B. R. Masters, M. V. Riley, J. Fischbarg, B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, noninvasive fluorometry and physiological correlates,” Exp. Eye Res. 36, 1–9 (1983).
[CrossRef]

B. R. Masters, S. Falk, B. Chance, “In vivo flavoprotein redox measurements of rabbit corneal normoxic–anoxic transitions,” Curr. Eye Res. 1, 623–627 (1982).
[CrossRef]

B. R. Masters, “In vitro corneal redox fluorometry,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 223–247.
[CrossRef]

B. R. Masters, “An optical method for the determination of oxygen concentration in the tear film,” in The Precorneal Tear Film in Health, Disease and Contact Lens Wear, F. Holly, ed. (Dry Eye Institute, Lubbock, Tex.1986), pp. 966–970.

B. R. Masters, “Noninvasive corneal redox fluorometry,” in Current Topics in Eye Research, J. Zadunaisky, H. Davson, eds. (Academic, London, 1984), pp. 139–200.

B. R. Masters, “Confocal microscopy of ocular tissue,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990, pp. 305–324.

B. R. Masters, “Effects of contact lenses on the oxygen concentration and epithelial mitochondrial redox state of rabbit cornea measured noninvasively with an 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. 2810–3860.

A. Kriete, B. R. Masters, “Three-dimensional visualization of the living cornea,” in Transactions of the Royal Microscopical Society, MICRO 90, V. Howard, ed. (Institute of Physics, Bristol, UK, 1990), Vol. 1, pp. 401–404.

B. R. Masters, G. S. Kino, “Confocal microscopy of the eye,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 152–171.
[CrossRef]

Montag, M.

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

Munakata, C.

S. Kimura, C. Munakata, “Depth resolution of the fluorescent confocal scanning optical microscope,” Appl. Opt. 29, 489–494 (1990).
[CrossRef] [PubMed]

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc. Am. 6, 1015–1019 (1989).
[CrossRef]

Paddock, S. W.

Peltzer, E. A.

S. D. Bennet, E. A. Peltzer, I. R. Smith, “Ultra-violet confocal metrology,” in Scanning Microscopy Technologies and Applications, E. C. Teague, ed., Proc. Soc. Photo-Opt. Instrum. Eng.897, 75–82 (1988).

Riley, M. V.

B. R. Masters, M. V. Riley, J. Fischbarg, B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, noninvasive fluorometry and physiological correlates,” Exp. Eye Res. 36, 1–9 (1983).
[CrossRef]

Smith, I. R.

S. D. Bennet, E. A. Peltzer, I. R. Smith, “Ultra-violet confocal metrology,” in Scanning Microscopy Technologies and Applications, E. C. Teague, ed., Proc. Soc. Photo-Opt. Instrum. Eng.897, 75–82 (1988).

Spring, H.

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

Thorell, B.

B. Chance, B. Thorell, “Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry,” J. Biol. Chem. 234, 3044–3050 (1959).
[PubMed]

Trendelenburg, M. F.

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

Urstadt, R.

Wilke, V.

H. G. Kapitza, V. Wilke, “Applications of the microscope system LSM,” in Scanning Imaging, T. Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 173–179 (1988).

Wilson, T.

T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

T. Wilson, “Optical aspects of confocal microscopy,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 93–141.

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]

Appl. Opt.

Biophys. J.

J. Eng, R. M. Lynch, R. S. Balaban, “Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes,” Biophys. J. 55, 621–630 (1989).
[CrossRef] [PubMed]

Cornea

M. A. Lemp, P. N. Dilly, A. Boyde, “Tandem-scanning (confocal) microscope for optically sectioning the living cornea,” Cornea 4, 205–209 (1986).

Curr. Eye Res.

B. R. Masters, “Noninvasive redox fluorometry: how light can be used to monitor alterations of corneal mitochondrial function,” Curr. Eye Res. 3, 23–26 (1984).
[CrossRef] [PubMed]

B. R. Masters, S. Falk, B. Chance, “In vivo flavoprotein redox measurements of rabbit corneal normoxic–anoxic transitions,” Curr. Eye Res. 1, 623–627 (1982).
[CrossRef]

Exp. Eye Res.

B. Chance, M. Lieberman, “Intrinsic fluorescence emission from the cornea at low temperatures: evidence of mitochondrial signals and their differing redox states in epithelial and endothelial sides,” Exp. Eye Res. 26, 111–117 (1978).
[CrossRef] [PubMed]

B. R. Masters, M. V. Riley, J. Fischbarg, B. Chance, “Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, noninvasive fluorometry and physiological correlates,” Exp. Eye Res. 36, 1–9 (1983).
[CrossRef]

J. Biol. Chem.

B. Chance, B. Thorell, “Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry,” J. Biol. Chem. 234, 3044–3050 (1959).
[PubMed]

J. Microsc.

B. R. Masters, S. W. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–275 (1990).
[CrossRef] [PubMed]

M. Montag, J. Kukulies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, H. Spring, “Working with the confocal UV-laser scanning microscope: specific DNA localization at high sensitivity and multiple parameter fluorescence,” J. Microsc. 163, 201–210 (1991).
[CrossRef] [PubMed]

T. Wilson, “Optical sectioning in confocal fluorescent microscopes,” J. Microsc. 154, 143–156 (1989).
[CrossRef]

J. Opt. Soc. Am.

S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc. Am. 6, 1015–1019 (1989).
[CrossRef]

Proc. Inst. Phys. (UK)

B. R. Masters, G. S. Kino, “Real-time confocal scanning imaging of the eye: instrument performance of reflection and fluorescence imaging,” Proc. Inst. Phys. (UK) 98, 625–628 (1989).

Scanning

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]

Other

A. Kriete, B. R. Masters, “Three-dimensional visualization of the living cornea,” in Transactions of the Royal Microscopical Society, MICRO 90, V. Howard, ed. (Institute of Physics, Bristol, UK, 1990), Vol. 1, pp. 401–404.

S. D. Bennet, E. A. Peltzer, I. R. Smith, “Ultra-violet confocal metrology,” in Scanning Microscopy Technologies and Applications, E. C. Teague, ed., Proc. Soc. Photo-Opt. Instrum. Eng.897, 75–82 (1988).

H. G. Kapitza, V. Wilke, “Applications of the microscope system LSM,” in Scanning Imaging, T. Wilson, ed., Proc. Soc. Photo-Opt. Instrum. Eng.1028, 173–179 (1988).

B. R. Masters, G. S. Kino, “Confocal microscopy of the eye,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 152–171.
[CrossRef]

B. R. Masters, “An optical method for the determination of oxygen concentration in the tear film,” in The Precorneal Tear Film in Health, Disease and Contact Lens Wear, F. Holly, ed. (Dry Eye Institute, Lubbock, Tex.1986), pp. 966–970.

T. Wilson, “Optical aspects of confocal microscopy,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990), pp. 93–141.

B. R. Masters, “In vitro corneal redox fluorometry,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990), pp. 223–247.
[CrossRef]

B. R. Masters, “Noninvasive corneal redox fluorometry,” in Current Topics in Eye Research, J. Zadunaisky, H. Davson, eds. (Academic, London, 1984), pp. 139–200.

B. R. Masters, “Confocal microscopy of ocular tissue,” in Confocal Microscopy, T. Wilson, ed. (Academic, London, 1990, pp. 305–324.

B. R. Masters, “Effects of contact lenses on the oxygen concentration and epithelial mitochondrial redox state of rabbit cornea measured noninvasively with an 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. 2810–3860.

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Figures (5)

Fig. 1
Fig. 1

Optical system of the Zeiss laser scanning microscope. The schematic laser beam paths of the laser confocal microscope are shown. The standard Zeiss laser scanning microscope has been fitted with two external lasers: an external argon-ion laser (488-nm, 514-nm laser lines) and an external UV argon-ion laser (a 364-nm line). There is an internal He–Ne laser (543- and 633-nm laser lines). Switchable mirrors and barrier filters permit UV fluorescence or reflectance measurements. The various barrier filters are located between the confocal variable pinhole and the beam splitter that is adjacent to the two photomultiplier detectors (PMT1, PMT2). For confocal UV operation the UV laser and an external beam expansion unit are added to the laser scanning confocal microscope. A Zeiss water objective of 25 ×, with an NA of 0.8, corrected for the UV, was used for fluorescence and reflected-mode confocal microscopy. FT, filters.

Fig. 2
Fig. 2

Z-scan confocal microscopic image of the anterior region of the cornea from an in vitro rabbit eye. The image is produced with backscattered light (488 nm). The uppermost bright region is the highly reflective superficial epithelial cells adjacent to the tear film. A bright line, 50 μm below, represents the highly reflective basal lamina. Directly above the basal lamina are the basal epithelial cells with their nuclei. Below the basal lamina are short horizontal lines that are the nuclei of the stromal fibroblasts. The 50-μm scale bar is shown.

Fig. 3
Fig. 3

Confocal microscopic image of the anterior region of the cornea from an in vitro rabbit eye. The image is produced with backscattered light (488 nm). The image plane is perpendicular to the optic axis of the eye. The nuclei of stromal fibroblasts cells are shown together with submicrometer filaments that interconnect the cells. The cell bodies are not visible.

Fig. 4
Fig. 4

(a) Confocal microscopic image of the anterior region of the cornea from an in vitro rabbit eye. The image is produced with backscattered light of 364 nm. The superficial epithelial cells and their nuclei are shown. (b) Similar focal plane view imaged with UV 364-nm light in the fluorescent mode. The image shows the fluorescence from the pyridine nucleotides in the corneal cells Emission is 400–500 nm.

Fig. 5
Fig. 5

(a) Confocal microscopic image of the endothelial cells of the cornea from an in vitro rabbit eye. The image is produced with backscattered light of 364 nm. (b) Confocal microscopic image of the endothelial cells of the cornea from an in vitro rabbit eye. The reduced pyridine nucleotide fluorescence is shown in the bright regions; the cell nuclei are the dark regions. The images are made through 400 μm of corneal tissue. The laser excitation was 364 nm, and the emission was 400–500 nm. The dark regions in the image are the nuclei of the endothelial cells. The bright regions show the fluorescence from the reduced pyridine nucleotides.

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