Abstract

A doublet contact element was added to a long-working-distance objective to increase the numerical aperture to 0.75 and to maintain the focus during in vivo examination of the eye. Optical sectioning by use of confocal slits permits visualization of weakly scattering structures within the cornea. With photographic film and a 1/60-s exposure time to limit the effect of eye movement, an effective optical section half-thickness of ~20 μm was realized. Structures observed in the cornea include epithelial cells (surface, wing, and basal cells), nerve-fiber bundles in the subepithelial region, keratocytes and inflammatory cells in the stroma, and endothelial cells.

© 1993 Optical Society of America

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References

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  1. C. J. Koester, “A comparison of various optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 19, pp. 207–214.
    [CrossRef]
  2. J. R. Benford, H. E. Rosenberger, “Microscope objectives and eyepieces,” in Handbook of Optics, W. G. Driscoll, W. Vaughn, eds. (McGraw-Hill, New York, 1978), Chap. 6, p. 3.
  3. W. S. Duke-Elder, Text-Book of Ophthalmology (Mosby, St. Louis, Mo., 1933), Vol. 1, p. 27, Fig. 53, used with permission of the publisher.
  4. D. M. Maurice, “Cellular membrane activity in the corneal endothelium of the intact eye,” Experientia 24, 1094–1095 (1968).
    [CrossRef] [PubMed]
  5. D. M. Maurice, “A scanning slit optical microscope,” Invest. Ophthalmol. Vis. Sci. 13, 1033–1037 (1974).
  6. R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, “In vivophotomicrography of the corneal endothelium,” Arch. Ophthalmol. 93, 143–145 (1975).
    [CrossRef] [PubMed]
  7. W. M. Bourne, B. E. McCarey, H. E. Kaufman, “Clinical specular microscopy,” Ophthalmology 81, 743–753 (1976).
  8. C. J. Koester, “Scanning mirror microscope with optical sectioning characteristics: applications in ophthalmology,” Appl. Opt. 19, 1749–1757 (1980).
    [CrossRef] [PubMed]
  9. B. R. Masters, S. Paddock, “In vitroconfocal imaging of the rabbit cornea,” J. Microsc. (Oxford) 158, 267–274 (1990).
    [CrossRef]
  10. 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).
    [CrossRef]
  11. H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
    [PubMed]
  12. M. Petráň, M. Hadravský, M. D. Egger, R. Galambos, “Tandem scanning reflected light microscope,”J. Opt. Soc. Am. 58, 661–664 (1968).
    [CrossRef]
  13. J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
    [CrossRef]
  14. T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 3, 72, and 74.
  15. L. E. Lohman, G. N. Rao, J. V. Aquavella, “The normal human corneal epithelium—in vivomicroscopic observations,” Arch. Ophthalmol. 100, 991–993 (1982).
    [CrossRef] [PubMed]
  16. S. L. Trokel, “Development of the excimer laser in ophthalmology,” Refract. Corneal Surg. 6, 357–362 (1990).
    [PubMed]
  17. E. S. Sherrard, R. J. Buckley, “Endothelial wrinkling: a complication of clinical specular microscopy,” in The Cornea in Health and Disease: Proceedings of the Sixth Congress of the European Society of Ophthalmologists, P. Trevor-Roper, ed. (Grune & Stratton, New York, 1981), pp. 67–74.
  18. oslo (optical system layout and optimization), Sinclair Optics, Fairport, N.Y. 14450.
  19. Cermax lamp LX150F, ILC Technology, Sunnyvale, Calif.
  20. C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).
  21. O. N. Serdarevic, C. J. Koester, “Colour wide field specular microscopic investigation of corneal surface disorders,” Trans. Ophthalmol. Soc. U.K. 104, 439–445 (1985).
  22. B. Schimmelpfennig, “Nerve structures in human central corneal epithelium,” Graefe’s Arch. Clin. Exp. Ophthalmol. 218, 14–20 (1982).
  23. American National Standard for the Safe Use of Lasers Z136.1-1986 (American National Standards Institute, Inc., New York, 1986).

1992 (1)

J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
[CrossRef]

1990 (4)

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

H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
[PubMed]

S. L. Trokel, “Development of the excimer laser in ophthalmology,” Refract. Corneal Surg. 6, 357–362 (1990).
[PubMed]

C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).

1985 (1)

O. N. Serdarevic, C. J. Koester, “Colour wide field specular microscopic investigation of corneal surface disorders,” Trans. Ophthalmol. Soc. U.K. 104, 439–445 (1985).

1982 (2)

B. Schimmelpfennig, “Nerve structures in human central corneal epithelium,” Graefe’s Arch. Clin. Exp. Ophthalmol. 218, 14–20 (1982).

L. E. Lohman, G. N. Rao, J. V. Aquavella, “The normal human corneal epithelium—in vivomicroscopic observations,” Arch. Ophthalmol. 100, 991–993 (1982).
[CrossRef] [PubMed]

1980 (1)

1976 (1)

W. M. Bourne, B. E. McCarey, H. E. Kaufman, “Clinical specular microscopy,” Ophthalmology 81, 743–753 (1976).

1975 (1)

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, “In vivophotomicrography of the corneal endothelium,” Arch. Ophthalmol. 93, 143–145 (1975).
[CrossRef] [PubMed]

1974 (1)

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

1968 (2)

D. M. Maurice, “Cellular membrane activity in the corneal endothelium of the intact eye,” Experientia 24, 1094–1095 (1968).
[CrossRef] [PubMed]

M. Petráň, M. Hadravský, M. D. Egger, R. Galambos, “Tandem scanning reflected light microscope,”J. Opt. Soc. Am. 58, 661–664 (1968).
[CrossRef]

Aquavella, J. V.

L. E. Lohman, G. N. Rao, J. V. Aquavella, “The normal human corneal epithelium—in vivomicroscopic observations,” Arch. Ophthalmol. 100, 991–993 (1982).
[CrossRef] [PubMed]

Benford, J. R.

J. R. Benford, H. E. Rosenberger, “Microscope objectives and eyepieces,” in Handbook of Optics, W. G. Driscoll, W. Vaughn, eds. (McGraw-Hill, New York, 1978), Chap. 6, p. 3.

Bourne, W. M.

W. M. Bourne, B. E. McCarey, H. E. Kaufman, “Clinical specular microscopy,” Ophthalmology 81, 743–753 (1976).

Buckley, R. J.

E. S. Sherrard, R. J. Buckley, “Endothelial wrinkling: a complication of clinical specular microscopy,” in The Cornea in Health and Disease: Proceedings of the Sixth Congress of the European Society of Ophthalmologists, P. Trevor-Roper, ed. (Grune & Stratton, New York, 1981), pp. 67–74.

Cavanagh, H. D.

J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
[CrossRef]

H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
[PubMed]

Duke-Elder, W. S.

W. S. Duke-Elder, Text-Book of Ophthalmology (Mosby, St. Louis, Mo., 1933), Vol. 1, p. 27, Fig. 53, used with permission of the publisher.

Egger, M. D.

Essepian, J.

H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
[PubMed]

Galambos, R.

Garana, R. M. R.

J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
[CrossRef]

Hadravský, M.

Jester, J. V.

J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
[CrossRef]

H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
[PubMed]

Kaufman, H. E.

W. M. Bourne, B. E. McCarey, H. E. Kaufman, “Clinical specular microscopy,” Ophthalmology 81, 743–753 (1976).

Kino, G. S.

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).
[CrossRef]

Koester, C. J.

C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).

O. N. Serdarevic, C. J. Koester, “Colour wide field specular microscopic investigation of corneal surface disorders,” Trans. Ophthalmol. Soc. U.K. 104, 439–445 (1985).

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

C. J. Koester, “A comparison of various optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 19, pp. 207–214.
[CrossRef]

Laing, R. A.

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, “In vivophotomicrography of the corneal endothelium,” Arch. Ophthalmol. 93, 143–145 (1975).
[CrossRef] [PubMed]

Leibowitz, H. M.

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, “In vivophotomicrography of the corneal endothelium,” Arch. Ophthalmol. 93, 143–145 (1975).
[CrossRef] [PubMed]

Lemp, M. A.

J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
[CrossRef]

H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
[PubMed]

Lohman, L. E.

L. E. Lohman, G. N. Rao, J. V. Aquavella, “The normal human corneal epithelium—in vivomicroscopic observations,” Arch. Ophthalmol. 100, 991–993 (1982).
[CrossRef] [PubMed]

Masters, B. R.

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

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).
[CrossRef]

Maurice, D. M.

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

D. M. Maurice, “Cellular membrane activity in the corneal endothelium of the intact eye,” Experientia 24, 1094–1095 (1968).
[CrossRef] [PubMed]

McCarey, B. E.

W. M. Bourne, B. E. McCarey, H. E. Kaufman, “Clinical specular microscopy,” Ophthalmology 81, 743–753 (1976).

Paddock, S.

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

Petrán, M.

Petroll, W. M.

J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
[CrossRef]

Rao, G. N.

L. E. Lohman, G. N. Rao, J. V. Aquavella, “The normal human corneal epithelium—in vivomicroscopic observations,” Arch. Ophthalmol. 100, 991–993 (1982).
[CrossRef] [PubMed]

Rosenberger, H. E.

J. R. Benford, H. E. Rosenberger, “Microscope objectives and eyepieces,” in Handbook of Optics, W. G. Driscoll, W. Vaughn, eds. (McGraw-Hill, New York, 1978), Chap. 6, p. 3.

Sandstrom, M. M.

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, “In vivophotomicrography of the corneal endothelium,” Arch. Ophthalmol. 93, 143–145 (1975).
[CrossRef] [PubMed]

Schimmelpfennig, B.

B. Schimmelpfennig, “Nerve structures in human central corneal epithelium,” Graefe’s Arch. Clin. Exp. Ophthalmol. 218, 14–20 (1982).

Serdarevic, O. N.

O. N. Serdarevic, C. J. Koester, “Colour wide field specular microscopic investigation of corneal surface disorders,” Trans. Ophthalmol. Soc. U.K. 104, 439–445 (1985).

Sheppard, C.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 3, 72, and 74.

Sherrard, E. S.

E. S. Sherrard, R. J. Buckley, “Endothelial wrinkling: a complication of clinical specular microscopy,” in The Cornea in Health and Disease: Proceedings of the Sixth Congress of the European Society of Ophthalmologists, P. Trevor-Roper, ed. (Grune & Stratton, New York, 1981), pp. 67–74.

Shields, W.

H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
[PubMed]

Trokel, S. L.

S. L. Trokel, “Development of the excimer laser in ophthalmology,” Refract. Corneal Surg. 6, 357–362 (1990).
[PubMed]

Wilson, T.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 3, 72, and 74.

Appl. Opt. (1)

Arch. Ophthalmol. (2)

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, “In vivophotomicrography of the corneal endothelium,” Arch. Ophthalmol. 93, 143–145 (1975).
[CrossRef] [PubMed]

L. E. Lohman, G. N. Rao, J. V. Aquavella, “The normal human corneal epithelium—in vivomicroscopic observations,” Arch. Ophthalmol. 100, 991–993 (1982).
[CrossRef] [PubMed]

CLAO J. (1)

H. D. Cavanagh, J. V. Jester, J. Essepian, W. Shields, M. A. Lemp, “Confocal microscopy of the living eye,” CLAO J. 16, 65–73 (1990).
[PubMed]

Experientia (1)

D. M. Maurice, “Cellular membrane activity in the corneal endothelium of the intact eye,” Experientia 24, 1094–1095 (1968).
[CrossRef] [PubMed]

Graefe’s Arch. Clin. Exp. Ophthalmol. (1)

B. Schimmelpfennig, “Nerve structures in human central corneal epithelium,” Graefe’s Arch. Clin. Exp. Ophthalmol. 218, 14–20 (1982).

Invest. Ophthalmol. Vis. Sci. (1)

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

J. Microsc. (Oxford) (2)

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

J. V. Jester, W. M. Petroll, R. M. R. Garana, M. A. Lemp, H. D. Cavanagh, “Comparison of in vivoand ex vivocellular structure in rabbit eyes detected by tandem scanning microscopy,” J. Microsc. (Oxford) 165, Pt. 1, 169–181 (1992).
[CrossRef]

J. Opt. Soc. Am. (1)

Ophthalmology (1)

W. M. Bourne, B. E. McCarey, H. E. Kaufman, “Clinical specular microscopy,” Ophthalmology 81, 743–753 (1976).

Refract. Corneal Surg. (1)

S. L. Trokel, “Development of the excimer laser in ophthalmology,” Refract. Corneal Surg. 6, 357–362 (1990).
[PubMed]

Trans. Ophthalmol. Soc. U.K. (1)

O. N. Serdarevic, C. J. Koester, “Colour wide field specular microscopic investigation of corneal surface disorders,” Trans. Ophthalmol. Soc. U.K. 104, 439–445 (1985).

Trans. R. Microsc. Soc. (1)

C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).

Other (9)

American National Standard for the Safe Use of Lasers Z136.1-1986 (American National Standards Institute, Inc., New York, 1986).

E. S. Sherrard, R. J. Buckley, “Endothelial wrinkling: a complication of clinical specular microscopy,” in The Cornea in Health and Disease: Proceedings of the Sixth Congress of the European Society of Ophthalmologists, P. Trevor-Roper, ed. (Grune & Stratton, New York, 1981), pp. 67–74.

oslo (optical system layout and optimization), Sinclair Optics, Fairport, N.Y. 14450.

Cermax lamp LX150F, ILC Technology, Sunnyvale, Calif.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), pp. 3, 72, and 74.

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).
[CrossRef]

C. J. Koester, “A comparison of various optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 19, pp. 207–214.
[CrossRef]

J. R. Benford, H. E. Rosenberger, “Microscope objectives and eyepieces,” in Handbook of Optics, W. G. Driscoll, W. Vaughn, eds. (McGraw-Hill, New York, 1978), Chap. 6, p. 3.

W. S. Duke-Elder, Text-Book of Ophthalmology (Mosby, St. Louis, Mo., 1933), Vol. 1, p. 27, Fig. 53, used with permission of the publisher.

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Figures (13)

Fig. 1
Fig. 1

Microscopic section of the cornea drawn diagrammatically. From the top the layers are e, epithelial cells; b, Bowman’s layer; s, stroma; d, Descemet’s membrane; and e, endothelial-cell layer. Within the stroma are seen collagen lamellae (gray) and keratocytes (black). The gaps between the lamellae are fixation artifacts (from Ref. 3).

Fig. 2
Fig. 2

Contact element in position relative to the objective lens. The front (contact) material is 1.05-mm-thick fused silica with a low-reflection coating designed for water immersion. The second and third glasses are Schott SFL6 and BK7, respectively. Radii are 13.94 and 5.98 mm, respectively.

Fig. 3
Fig. 3

Second configuration of the contact element, designed for the posterior cornea. The fused-silica front-plate thickness has been decreased to 0.6 mm to compensate for the increased thickness of corneal tissue. Other components are the same glasses and have the same radii and thicknesses as in Fig. 2. The focusing mechanism is shown in cross section. S, the mount for the contact element, slides on bearing surface B, which is fixed to the outer surface of objective O. The compression spring, P, holds the lens mount, S, against the focusing collar, H. Focusing is accomplished by turning H on the threads shown schematically at T.

Fig. 4
Fig. 4

Layout of the scanning slit confocal microscope. The light source at A is a 150-W xenon arc lamp,19 the output of which is a collimated beam. Lens L1 brings the image of the arc to focus at slit S1, where the distribution is relatively uniform over a 6-mm diameter, equal to the length of the slit. Lens L2 produces a virtual image of slit S1 that is located at the normal-image plane of objective L3. This virtual image is confocal with slit S2. Incident light passes through the upper half of objective lens L3 and through the contact element to form an image of S1 at focal plane F. The oscillating rotational motion of mirror M2 causes the slit image to scan across the focal plane. Light reflected from the illuminated region of the specimen is transmitted through the lower half of the objective, is descanned by the motion of mirror M2, and is focused at S2. The field lens, L4, images the objective aperture at the third facet of mirror M2. The third reflection from scanning mirror M2 causes the image of S2 to scan across the final image plane and to lay down the image in a continuous fashion. For photographic exposure the lamp is pulsed to 500 W for the duration of the shutter opening, typically 1/60 s. This increases the illuminance at S1 by a factor of ~7 for the duration of the pulse. Mirrors M3–M6 direct the image beam through slit S2, back to mirror M2, and finally to the image plane.

Fig. 5
Fig. 5

Enlarged view of the focal plane and adjacent volume. The focal diamond (hatched area) is the only region in which light from the illumination bundle can be scattered or reflected directly into the imaging bundle. Illumination rays all lie between the limiting rays that are shown as lines with single arrowheads. Imaging rays are bounded by the rays with double arrowheads. Dashed lines indicate the region beyond the focal plane that can contribute light to the image. The minimum angle A of the rays in the illumination and imaging light bundles is determined by width D of the opaque divider and the focal length of objective lens O. The half-thickness of the focal diamond is h.

Fig. 6
Fig. 6

Comparison of (a) the geometrical focal diamond and (b) a plot of stray light that reaches the image from an out-of-focus reflecting plane located at a distance x from focus. At point h′ the illuminance has fallen to a value of one tenth that for an in-focus plane (x = 0). The width w of the image of slit S1 is 12 μm. The slit widths for these calculations are S2, 0.5 mm; S1, 0.17 mm; opaque divider, 2.5 mm. In this case h = 38 μm and h′ = 21 μm.

Fig. 7
Fig. 7

Basal epithelial cells of a 62-year-old subject with normal corneas. Bar, 100 μm.

Fig. 8
Fig. 8

Nerves located at the interface between the basal epithelial cells and Bowman’s layer. The orientation of the nerves is radial, with the pupil located above and to the right of this photomicrograph. Bar, 100 μm.

Fig. 9
Fig. 9

Keratocytes in the stroma and a pressure-induced dark band. Bar, 100 μm.

Fig. 10
Fig. 10

Endothelial cells and guttata (dark regions). Bar, 100 μm.

Fig. 11
Fig. 11

Serial optical sections in the posterior cornea. (a) Focus at the endothelial-cell layer. (b) Focus 7 μm anterior to the endothelium; out-of-focus endothelial cells and out-of-focus keratocytes are seen. (c) Focus 18 μm anterior to the endothelium, which is no longer distinguishable. K in (b) and (c) denotes the same keratocyte in these two photomicrographs. Bar, 100 μm.

Fig. 12
Fig. 12

Ray-traced simulation of blur patterns formed at the image plane in a full-aperture system, specifically the conventional circular-aperture (pinhole) confocal system. The source is a point source (either self-luminous or scattering) that is located on the axis at 10, 20, 30, and 40 μm from the focal plane. The ray tracing simulates an objective with 0.75 NA and an index of the medium that is equal to 1.376 (the cornea). The circle represents an aperture in the image plane of 0.5-mm diameter to match the width of the slit in Fig. 13 below. As the point source is moved farther from the focal plane the blur pattern becomes larger, as expected. In (d), at an out-of-focus distance of 40 μm, a portion of the light is still passing through the circular aperture.

Fig. 13
Fig. 13

Ray-traced simulation of blur patterns formed at the image plane in a divided-aperture system. As in Fig. 12, the source is a point source located on the axis, and the NA and index of refraction are the same. A central divider (as illustrated in Fig. 5) occupies 0.28 of the aperture diameter. The vertical lines represent the edges of a slit, located in the image plane, that has a width of 0.5 mm. When the point source is 40 μm from the focal plane in (d), the entire blur pattern falls outside the slit and would therefore contribute nothing to the image.

Equations (2)

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

h = w / ( 2 tan A ) ,
depth of focus = ± n λ / [ 2 ( NA ) 2 ] ,

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