Abstract

Three-dimensional confocal microscopy of a living in situ rabbit cornea, in a freshly excised eye, is demonstrated in two movie loops. A specimen chamber for the eye was designed and constructed to maintain the unstained, unfixed, in situ cornea in a viable physiological state during data acquisition. The 400 micron thick, transparent, cornea has been optically sectioned into 365 sections using a laser scanning confocal microscope. A high numerical aperture, water immersion microscope objective minimized the spherical aberrations which would occur with the use of an oil immersion objective. Depth dependent light attenuation due to absorption and scatter within the specimen was manually compensated at each sampled section. Isometric sampling resulted in near-cubic voxels which compensated for the reduced microscopic resolution in the z-axis as compared to x- and y- resolution.

© 1998 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. B. R. Masters, Selected Papers on Confocal Microscopy, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1996).
  2. B. R. Masters, “Confocal Microscopy of Ocular Tissue,” in Confocal Microscopy,T. Wilson, ed. (Academic Press, London, 1990), 305–324.
  3. B. R. Masters and G. S. Kino, “Confocal Microscopy of the Eye,” in Noninvasive Diagnostic Techniques in Ophthalmology,B. R. Masters ed. (Springer-Verlag, New York, 1990), 152–171.
    [Crossref]
  4. B. R. Masters, G. Gonnord, and P. Corcuff, “Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope,” J. Microsc. 185, 329–338 (1997).
    [Crossref] [PubMed]
  5. H. E. Keller, “Objective lenses for confocal microscopy,” in The Handbook of Biological Confocal Microscopy, 2 ed.,J. B. Pawley, ed. (Plenum Press, New York, 1995), 111–126.
  6. M. Levoy, “Display of Surfaces from Volume Data,” PhD dissertation, (University of North Carolina, Chapel Hill, N.C.1989)
  7. N. S. White, “Visualization Systems for Multidimensional CLSM Images,” in Handbook of Biological Confocal Microscopy,J. B. Pawley, ed. (Plenum Press, New York1990), 211–254.
  8. H. T. M. Van der Voort, G. J. Brankenhoff, and M. W. Baarlslag, “Three-dimensiona visualization methods for confocal microscopy,” J. Microsc. 153, 123–132 (1989).
    [Crossref] [PubMed]
  9. H. T. M. van der Voort and A. W. M. Smeudlers, “Acquisition, Processing, and Visualization of Three-Dimensional Confocal Images,” in Electronic Light Microscopy,D. Shotton, ed. (Wiley-Liss, New York, 1992), 247–271.
  10. A. Kriete, Visualization in Biomedical Microscopies, (VCH, New York, 1992).
  11. M. J. Cookson, R. A. Reynolds, and D-C. Abrams, “Three-dimensional visualization,” in Image analysis in histology, (R. Wootton, D. R. Springall, and J. M. Polak, eds. (Cambridge University Press, Cambridge, UK, 1995), 313–335.
  12. O. Minet, G. Müller, and J. Beuthan, Selected Papers on Optical Tomography, Fundamentals and Applications in Medicine, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1998).
  13. R. L. McCally and R. A. Farrell, “Light scattering from cornea and corneal transparency,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer Verlag, New York, 1990), 189–210.
    [Crossref]
  14. V. Tuchin, Selected Papers on Tissue Optics, Applications in Medical Diagnostics and Therapy, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1994).
  15. M. A. Lemp, P. N. Dilly, and A. Boyde, “Tandem-scanning (confocal) microscopy of the full-thickness cornea,” Cornea 4, 205–209 (1986).
  16. B. R. Masters and S. W. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–274 (1990).
    [Crossref] [PubMed]
  17. A. Kriete and B. R. Masters, “Three-dimensional visualization of the living cornea,” MICRO 90, 1, (Royal Microscopical Society, Oxford, 1990), 401–404.
  18. B. R. Masters and 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]
  19. B. R. Masters, “Two and three-dimensional visualization of the living cornea and ocular lens,” Machine Vision and Applications 4, 227–232 (1991).
    [Crossref]
  20. G. Q. Xiao, G. S. Kino, and 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]
  21. B. R. Masters and G. S. Kino, “Charge-coupled devices for quantitative Nipkow disk real-time scanning confocal microscopy,” in Electronic Light Microscopy: The principles and practice of intensified fluorescence, video-enhanced contrast and confocal scanning optical microscopy,D. Shotton, ed. (Wiley/Liss, Inc., New York, 1993), 315–327.
  22. B. R. Masters, “Two- and Three-Dimensional Confocal Microscopy of the Eye,” in Multidimensional Microscopy,P. C. Cheng, T. H. Lin, W. L. Wu, and J. L. Wu, eds. (Springer-Verlag, New York, 1993), 321–338.
  23. B. R. Masters and M. A. Farmer, “Three-dimensional confocal microscopy and visualization of the in situ cornea,” Comput. Med. Imag. Graph. 17, 211–219 (1993).
    [Crossref]
  24. B. R. Masters, “Specimen preparation and chamber for confocal microscopy of the eye,” Scanning Microscopy 7, 645–651 (1993).
    [PubMed]
  25. B. R. Masters and A. A. Thaer, “Real-time scanning slit confocal microscopy of the in vivo human cornea,” Appl. Opt. 33, 695–701 (1994).
    [Crossref] [PubMed]
  26. B.R. Masters, “Scanning Slit Confocal Microscopy of the in vivo Cornea,” Opt. Eng. 34, 684–692 (1995).
    [Crossref]

1997 (1)

B. R. Masters, G. Gonnord, and P. Corcuff, “Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope,” J. Microsc. 185, 329–338 (1997).
[Crossref] [PubMed]

1995 (1)

B.R. Masters, “Scanning Slit Confocal Microscopy of the in vivo Cornea,” Opt. Eng. 34, 684–692 (1995).
[Crossref]

1994 (1)

1993 (2)

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

B. R. Masters, “Specimen preparation and chamber for confocal microscopy of the eye,” Scanning Microscopy 7, 645–651 (1993).
[PubMed]

1991 (1)

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

1990 (4)

G. Q. Xiao, G. S. Kino, and 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 and S. W. Paddock, “In vitro confocal imaging of the rabbit cornea,” J. Microsc. 158, 267–274 (1990).
[Crossref] [PubMed]

A. Kriete and B. R. Masters, “Three-dimensional visualization of the living cornea,” MICRO 90, 1, (Royal Microscopical Society, Oxford, 1990), 401–404.

B. R. Masters and 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]

1989 (1)

H. T. M. Van der Voort, G. J. Brankenhoff, and M. W. Baarlslag, “Three-dimensiona visualization methods for confocal microscopy,” J. Microsc. 153, 123–132 (1989).
[Crossref] [PubMed]

1986 (1)

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

Abrams, D-C.

M. J. Cookson, R. A. Reynolds, and D-C. Abrams, “Three-dimensional visualization,” in Image analysis in histology, (R. Wootton, D. R. Springall, and J. M. Polak, eds. (Cambridge University Press, Cambridge, UK, 1995), 313–335.

Baarlslag, M. W.

H. T. M. Van der Voort, G. J. Brankenhoff, and M. W. Baarlslag, “Three-dimensiona visualization methods for confocal microscopy,” J. Microsc. 153, 123–132 (1989).
[Crossref] [PubMed]

Beuthan, J.

O. Minet, G. Müller, and J. Beuthan, Selected Papers on Optical Tomography, Fundamentals and Applications in Medicine, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1998).

Boyde, A.

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

Brankenhoff, G. J.

H. T. M. Van der Voort, G. J. Brankenhoff, and M. W. Baarlslag, “Three-dimensiona visualization methods for confocal microscopy,” J. Microsc. 153, 123–132 (1989).
[Crossref] [PubMed]

Cookson, M. J.

M. J. Cookson, R. A. Reynolds, and D-C. Abrams, “Three-dimensional visualization,” in Image analysis in histology, (R. Wootton, D. R. Springall, and J. M. Polak, eds. (Cambridge University Press, Cambridge, UK, 1995), 313–335.

Corcuff, P.

B. R. Masters, G. Gonnord, and P. Corcuff, “Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope,” J. Microsc. 185, 329–338 (1997).
[Crossref] [PubMed]

Dilly, P. N.

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

Farmer, M. A.

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

Farrell, R. A.

R. L. McCally and R. A. Farrell, “Light scattering from cornea and corneal transparency,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer Verlag, New York, 1990), 189–210.
[Crossref]

Gonnord, G.

B. R. Masters, G. Gonnord, and P. Corcuff, “Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope,” J. Microsc. 185, 329–338 (1997).
[Crossref] [PubMed]

Keller, H. E.

H. E. Keller, “Objective lenses for confocal microscopy,” in The Handbook of Biological Confocal Microscopy, 2 ed.,J. B. Pawley, ed. (Plenum Press, New York, 1995), 111–126.

Kino, G. S.

G. Q. Xiao, G. S. Kino, and 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 and G. S. Kino, “Charge-coupled devices for quantitative Nipkow disk real-time scanning confocal microscopy,” in Electronic Light Microscopy: The principles and practice of intensified fluorescence, video-enhanced contrast and confocal scanning optical microscopy,D. Shotton, ed. (Wiley/Liss, Inc., New York, 1993), 315–327.

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

Kriete, A.

A. Kriete and B. R. Masters, “Three-dimensional visualization of the living cornea,” MICRO 90, 1, (Royal Microscopical Society, Oxford, 1990), 401–404.

A. Kriete, Visualization in Biomedical Microscopies, (VCH, New York, 1992).

Lemp, M. A.

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

Levoy, M.

M. Levoy, “Display of Surfaces from Volume Data,” PhD dissertation, (University of North Carolina, Chapel Hill, N.C.1989)

Masters, B. R.

B. R. Masters, G. Gonnord, and P. Corcuff, “Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope,” J. Microsc. 185, 329–338 (1997).
[Crossref] [PubMed]

B. R. Masters and A. A. Thaer, “Real-time scanning slit confocal microscopy of the in vivo human cornea,” Appl. Opt. 33, 695–701 (1994).
[Crossref] [PubMed]

B. R. Masters, “Specimen preparation and chamber for confocal microscopy of the eye,” Scanning Microscopy 7, 645–651 (1993).
[PubMed]

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

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

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

A. Kriete and B. R. Masters, “Three-dimensional visualization of the living cornea,” MICRO 90, 1, (Royal Microscopical Society, Oxford, 1990), 401–404.

B. R. Masters and 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, and 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 and G. S. Kino, “Charge-coupled devices for quantitative Nipkow disk real-time scanning confocal microscopy,” in Electronic Light Microscopy: The principles and practice of intensified fluorescence, video-enhanced contrast and confocal scanning optical microscopy,D. Shotton, ed. (Wiley/Liss, Inc., New York, 1993), 315–327.

B. R. Masters, “Two- and Three-Dimensional Confocal Microscopy of the Eye,” in Multidimensional Microscopy,P. C. Cheng, T. H. Lin, W. L. Wu, and J. L. Wu, eds. (Springer-Verlag, New York, 1993), 321–338.

B. R. Masters, Selected Papers on Confocal Microscopy, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1996).

B. R. Masters, “Confocal Microscopy of Ocular Tissue,” in Confocal Microscopy,T. Wilson, ed. (Academic Press, London, 1990), 305–324.

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

Masters, B.R.

B.R. Masters, “Scanning Slit Confocal Microscopy of the in vivo Cornea,” Opt. Eng. 34, 684–692 (1995).
[Crossref]

McCally, R. L.

R. L. McCally and R. A. Farrell, “Light scattering from cornea and corneal transparency,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer Verlag, New York, 1990), 189–210.
[Crossref]

Minet, O.

O. Minet, G. Müller, and J. Beuthan, Selected Papers on Optical Tomography, Fundamentals and Applications in Medicine, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1998).

Müller, G.

O. Minet, G. Müller, and J. Beuthan, Selected Papers on Optical Tomography, Fundamentals and Applications in Medicine, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1998).

Paddock, S. W.

Reynolds, R. A.

M. J. Cookson, R. A. Reynolds, and D-C. Abrams, “Three-dimensional visualization,” in Image analysis in histology, (R. Wootton, D. R. Springall, and J. M. Polak, eds. (Cambridge University Press, Cambridge, UK, 1995), 313–335.

Smeudlers, A. W. M.

H. T. M. van der Voort and A. W. M. Smeudlers, “Acquisition, Processing, and Visualization of Three-Dimensional Confocal Images,” in Electronic Light Microscopy,D. Shotton, ed. (Wiley-Liss, New York, 1992), 247–271.

Thaer, A. A.

Tuchin, V.

V. Tuchin, Selected Papers on Tissue Optics, Applications in Medical Diagnostics and Therapy, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1994).

Voort, H. T. M. Van der

H. T. M. Van der Voort, G. J. Brankenhoff, and M. W. Baarlslag, “Three-dimensiona visualization methods for confocal microscopy,” J. Microsc. 153, 123–132 (1989).
[Crossref] [PubMed]

H. T. M. van der Voort and A. W. M. Smeudlers, “Acquisition, Processing, and Visualization of Three-Dimensional Confocal Images,” in Electronic Light Microscopy,D. Shotton, ed. (Wiley-Liss, New York, 1992), 247–271.

White, N. S.

N. S. White, “Visualization Systems for Multidimensional CLSM Images,” in Handbook of Biological Confocal Microscopy,J. B. Pawley, ed. (Plenum Press, New York1990), 211–254.

Xiao, G. Q.

G. Q. Xiao, G. S. Kino, and 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. (2)

Comput. Med. Imag. Graph. (1)

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

Cornea (1)

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

J. Microsc. (3)

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

B. R. Masters, G. Gonnord, and P. Corcuff, “Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope,” J. Microsc. 185, 329–338 (1997).
[Crossref] [PubMed]

H. T. M. Van der Voort, G. J. Brankenhoff, and M. W. Baarlslag, “Three-dimensiona visualization methods for confocal microscopy,” J. Microsc. 153, 123–132 (1989).
[Crossref] [PubMed]

Machine Vision and Applications (1)

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

MICRO 90, (1)

A. Kriete and B. R. Masters, “Three-dimensional visualization of the living cornea,” MICRO 90, 1, (Royal Microscopical Society, Oxford, 1990), 401–404.

Opt. Eng. (1)

B.R. Masters, “Scanning Slit Confocal Microscopy of the in vivo Cornea,” Opt. Eng. 34, 684–692 (1995).
[Crossref]

Scanning Microscopy (1)

B. R. Masters, “Specimen preparation and chamber for confocal microscopy of the eye,” Scanning Microscopy 7, 645–651 (1993).
[PubMed]

Scanning, (1)

G. Q. Xiao, G. S. Kino, and 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 (14)

B. R. Masters and G. S. Kino, “Charge-coupled devices for quantitative Nipkow disk real-time scanning confocal microscopy,” in Electronic Light Microscopy: The principles and practice of intensified fluorescence, video-enhanced contrast and confocal scanning optical microscopy,D. Shotton, ed. (Wiley/Liss, Inc., New York, 1993), 315–327.

B. R. Masters, “Two- and Three-Dimensional Confocal Microscopy of the Eye,” in Multidimensional Microscopy,P. C. Cheng, T. H. Lin, W. L. Wu, and J. L. Wu, eds. (Springer-Verlag, New York, 1993), 321–338.

B. R. Masters, Selected Papers on Confocal Microscopy, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1996).

B. R. Masters, “Confocal Microscopy of Ocular Tissue,” in Confocal Microscopy,T. Wilson, ed. (Academic Press, London, 1990), 305–324.

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

H. T. M. van der Voort and A. W. M. Smeudlers, “Acquisition, Processing, and Visualization of Three-Dimensional Confocal Images,” in Electronic Light Microscopy,D. Shotton, ed. (Wiley-Liss, New York, 1992), 247–271.

A. Kriete, Visualization in Biomedical Microscopies, (VCH, New York, 1992).

M. J. Cookson, R. A. Reynolds, and D-C. Abrams, “Three-dimensional visualization,” in Image analysis in histology, (R. Wootton, D. R. Springall, and J. M. Polak, eds. (Cambridge University Press, Cambridge, UK, 1995), 313–335.

O. Minet, G. Müller, and J. Beuthan, Selected Papers on Optical Tomography, Fundamentals and Applications in Medicine, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1998).

R. L. McCally and R. A. Farrell, “Light scattering from cornea and corneal transparency,” in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer Verlag, New York, 1990), 189–210.
[Crossref]

V. Tuchin, Selected Papers on Tissue Optics, Applications in Medical Diagnostics and Therapy, (SPIE, The International Society for Optical Engineering, Bellingham, WA, 1994).

H. E. Keller, “Objective lenses for confocal microscopy,” in The Handbook of Biological Confocal Microscopy, 2 ed.,J. B. Pawley, ed. (Plenum Press, New York, 1995), 111–126.

M. Levoy, “Display of Surfaces from Volume Data,” PhD dissertation, (University of North Carolina, Chapel Hill, N.C.1989)

N. S. White, “Visualization Systems for Multidimensional CLSM Images,” in Handbook of Biological Confocal Microscopy,J. B. Pawley, ed. (Plenum Press, New York1990), 211–254.

Supplementary Material (2)

» Media 1: MOV (4857 KB)     
» Media 2: MOV (4700 KB)     

Cited By

OSA participates in Crossref's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (2)

Fig. 1.
Fig. 1.

Three-dimensional living rabbit cornea (full thickness 400 microns). [Media 1]

Fig. 2.
Fig. 2.

Keratocyte nuclei in the posterior stroma (high resolution, partial thickness, 5 microns). This reconstructed volume of the stroma is near the posterior endothelial cell layer of the cornea. [Media 2]

Metrics