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.

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References

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  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, 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, 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 York 1990), 211-254.
  8. H. T. M. Van der Voort, G. J. Brankenhoff, M. W. Baarlslag, "Three-dimensional visualization methods for confocal microscopy," J. Microsc. 153, 123-132 (1989).
    [CrossRef] [PubMed]
  9. H. T. M. van der Voort, 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, D-C. Abrams, "Three-dimensional visualization," in Image analysis in histology, (R. Wootton, D. R. Springall, J. M. Polak, eds. (Cambridge University Press, Cambridge, UK, 1995), 313-335.
  12. O. Minet, G. M?ller, 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, 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, A. Boyde, "Tandem-scanning (confocal) microscopy of the full- thickness cornea," Cornea 4, 205-209 (1986).
  16. B. R. Masters, S. W. Paddock, "In vitro confocal imaging of the rabbit cornea," J. Microsc. 158, 267-274 (1990).
    [CrossRef] [PubMed]
  17. A. Kriete, B. R. Masters, "Three-dimensional visualization of the living cornea," MICRO 90, 1, (Royal Microscopical Society, Oxford, 1990), 401- 404.
  18. 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]
  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, 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, 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, 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, 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]

Other (26)

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, 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]

B. R. Masters, G. Gonnord, 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. 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 York 1990), 211-254.

H. T. M. Van der Voort, G. J. Brankenhoff, M. W. Baarlslag, "Three-dimensional visualization methods for confocal microscopy," J. Microsc. 153, 123-132 (1989).
[CrossRef] [PubMed]

H. T. M. van der Voort, 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, D-C. Abrams, "Three-dimensional visualization," in Image analysis in histology, (R. Wootton, D. R. Springall, J. M. Polak, eds. (Cambridge University Press, Cambridge, UK, 1995), 313-335.

O. Minet, G. M?ller, 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, 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).

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

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

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

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, "Two and three-dimensional visualization of the living cornea and ocular lens," Machine Vision and Applications 4, 227-232 (1991).
[CrossRef]

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, "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, 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]

B. R. Masters, 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, "Scanning Slit Confocal Microscopy of the in vivo Cornea," Opt. Eng. 34, 684-692 (1995).
[CrossRef]

Supplementary Material (2)

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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]

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