Abstract

Optical sectioning is the simultaneous illumination and viewing of only a thin region of a specimen. An illuminated slit is imaged at the plane of interest and is swept laterally by the action of an oscillating mirror. The light returning from the specimen reflects from a second facet of the oscillating mirror and forms a stationary image of the illuminated slit. At this stationary image a second slit is placed, which passes light from the desired plane and rejects scattered light from other depths within the specimen. Light passing through the second slit is reflected from the third facet of the oscillating mirror and is focused to the final image plane. The image is reconstructed as the image of the second slit sweeps across the image plane. An important ophthalmological application is the examination of the endothelial cell layer of the cornea, either by contact or noncontact techniques. Optimization for image illuminance and resolution is discussed.

© 1980 Optical Society of America

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References

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  1. A. Vogt, Graefes Arch. Ophthalmol. 101, 123 (1920).
    [CrossRef]
  2. D. Maurice, Experientia 24, 1094 (1968).
    [CrossRef] [PubMed]
  3. R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, Arch. Ophthalmol. 93, 143 (1975).
    [CrossRef] [PubMed]
  4. W. M. Bourne, H. E. Kaufman, Am. J. Ophthalmol. 81, 319 (1976).
    [PubMed]
  5. W. M. Bourne, B. E. McCarey, H. E. Kaufman, Trans. Am. Acad. Ophthalmol. Otolaryngol. 81, OP-743 (1976).
  6. D. Maurice, Invest. Ophthalmol. 13, 1033 (1974).
    [PubMed]
  7. S. C. Baer, U.S. Patent3,547,512 (1970).
  8. P. Davidovits, M. D. Egger, Appl. Opt. 10, 1615 (1971).
    [CrossRef] [PubMed]
  9. M. Petran, M. Hadravsky, M. D. Egger, R. Galambos, J. Opt. Soc. Am. 58, 661 (1968).
    [CrossRef]
  10. Electronics Division, Bulova Watch Co., Inc., Woodside, N.Y. 11377.
  11. A. C. Hardy, F. H. Perrin, The Principles of Optics (McGraw-Hill, New York, 1932), pp. 510–513.

1976

W. M. Bourne, H. E. Kaufman, Am. J. Ophthalmol. 81, 319 (1976).
[PubMed]

W. M. Bourne, B. E. McCarey, H. E. Kaufman, Trans. Am. Acad. Ophthalmol. Otolaryngol. 81, OP-743 (1976).

1975

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, Arch. Ophthalmol. 93, 143 (1975).
[CrossRef] [PubMed]

1974

D. Maurice, Invest. Ophthalmol. 13, 1033 (1974).
[PubMed]

1971

1968

1920

A. Vogt, Graefes Arch. Ophthalmol. 101, 123 (1920).
[CrossRef]

Baer, S. C.

S. C. Baer, U.S. Patent3,547,512 (1970).

Bourne, W. M.

W. M. Bourne, H. E. Kaufman, Am. J. Ophthalmol. 81, 319 (1976).
[PubMed]

W. M. Bourne, B. E. McCarey, H. E. Kaufman, Trans. Am. Acad. Ophthalmol. Otolaryngol. 81, OP-743 (1976).

Davidovits, P.

Egger, M. D.

Galambos, R.

Hadravsky, M.

Hardy, A. C.

A. C. Hardy, F. H. Perrin, The Principles of Optics (McGraw-Hill, New York, 1932), pp. 510–513.

Kaufman, H. E.

W. M. Bourne, B. E. McCarey, H. E. Kaufman, Trans. Am. Acad. Ophthalmol. Otolaryngol. 81, OP-743 (1976).

W. M. Bourne, H. E. Kaufman, Am. J. Ophthalmol. 81, 319 (1976).
[PubMed]

Laing, R. A.

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, Arch. Ophthalmol. 93, 143 (1975).
[CrossRef] [PubMed]

Leibowitz, H. M.

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, Arch. Ophthalmol. 93, 143 (1975).
[CrossRef] [PubMed]

Maurice, D.

D. Maurice, Invest. Ophthalmol. 13, 1033 (1974).
[PubMed]

D. Maurice, Experientia 24, 1094 (1968).
[CrossRef] [PubMed]

McCarey, B. E.

W. M. Bourne, B. E. McCarey, H. E. Kaufman, Trans. Am. Acad. Ophthalmol. Otolaryngol. 81, OP-743 (1976).

Perrin, F. H.

A. C. Hardy, F. H. Perrin, The Principles of Optics (McGraw-Hill, New York, 1932), pp. 510–513.

Petran, M.

Sandstrom, M. M.

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, Arch. Ophthalmol. 93, 143 (1975).
[CrossRef] [PubMed]

Vogt, A.

A. Vogt, Graefes Arch. Ophthalmol. 101, 123 (1920).
[CrossRef]

Am. J. Ophthalmol.

W. M. Bourne, H. E. Kaufman, Am. J. Ophthalmol. 81, 319 (1976).
[PubMed]

Appl. Opt.

Arch. Ophthalmol.

R. A. Laing, M. M. Sandstrom, H. M. Leibowitz, Arch. Ophthalmol. 93, 143 (1975).
[CrossRef] [PubMed]

Experientia

D. Maurice, Experientia 24, 1094 (1968).
[CrossRef] [PubMed]

Graefes Arch. Ophthalmol.

A. Vogt, Graefes Arch. Ophthalmol. 101, 123 (1920).
[CrossRef]

Invest. Ophthalmol.

D. Maurice, Invest. Ophthalmol. 13, 1033 (1974).
[PubMed]

J. Opt. Soc. Am.

Trans. Am. Acad. Ophthalmol. Otolaryngol.

W. M. Bourne, B. E. McCarey, H. E. Kaufman, Trans. Am. Acad. Ophthalmol. Otolaryngol. 81, OP-743 (1976).

Other

Electronics Division, Bulova Watch Co., Inc., Woodside, N.Y. 11377.

A. C. Hardy, F. H. Perrin, The Principles of Optics (McGraw-Hill, New York, 1932), pp. 510–513.

S. C. Baer, U.S. Patent3,547,512 (1970).

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Figures (12)

Fig. 1
Fig. 1

Cross section of the cornea, not to scale: T, tear film; Ep, epithelial cell layer; B, Bowman's membrane; S, stroma; D, Descemet's membrane; En, endothelial cell layer; A, aqueous.

Fig. 2
Fig. 2

Divided aperture objective as used by Maurice.2

Fig. 3
Fig. 3

Schematic diagram of scanning mirror microscope: L1, condenser; S1, first slit; L2, collimator; A, rectangular aperture; M1, oscillating mirror; L3, microscope objective; C, cornea; L4, telescope lens having the same focal length as L2; L5, field lens; S2, second slit; M2, stationary mirror; L6, image-forming lens; F, film plane or eyepiece image plane.

Fig. 4
Fig. 4

Ray diagram illustrating optical sectioning characteristics: θn, angle of the illumination ray with the minimum inclination; S, image of slit S1 (the width of the slit image at S is w); 2h, thickness of the optical section; A, a plane outside the optical section; B′ and B, planes at the boundary of the optical section; C, a plane within the optical section. Downward-pointing arrows indicate illumination rays; upward-pointing double arrows indicate image-forming rays. The rays shown are at the edges of the illumination and image-forming bundles. The crosshatched area indicates the region of the specimen in which any light scattered in the direction of the imaging aperture can pass through slit S2 and reach the image.

Fig. 5
Fig. 5

Image illuminance as a function of focus. 20× objective, water immersed to a specularly reflecting surface, no central aperture stop.

Fig. 6
Fig. 6

Partial diagram of scanning mirror microscope with opaque central aperture stop. Symbols are the same as in Fig. 3.

Fig. 7
Fig. 7

Optical sectioning effect in the case of noncontact endothelial microscopy. Optical section half-thickness h is made less than the thickness of the cornea so that the air/tear layer surface is outside the optical section.

Fig. 8
Fig. 8

Rabbit endothelium photographed with the scanning mirror microscope using a 20× Nikon dipping cone objective. Slit width of ∼60 μm at the specimen; slit direction horizontal; scan direction vertical. The image dimensions are ∼950 × 600 μm, an area of 0.57 mm2.

Fig. 9
Fig. 9

Endothelial image obtained with a conventional specular microscope. Size of field of cells is ∼130 × 370 μm.

Fig. 10
Fig. 10

Serial photomicrographs of a rabbit cornea taken with a 40× N.A. 0.75 objective. A reflection artifact is seen in all four images. (a) microscope focused on the endothelial layer; (b) microscope focused 25 μm anterior to the endothelium using the graduated fine focus adjustment; (c) 100 μm anterior to the endothelium; (d) 370 μm anterior to the endothelium. The cells seen in d are epithelial cells. Field size is ∼350 × 280 μm.

Fig. 11
Fig. 11

Noncontact images of a rabbit cornea: (a) air/tear layer surface with 1/125-sec exposure on ASA 800 film; (b) endothelial cell layer with 0.5-sec exposure on same film. Total field size is ∼760 × 1000 μm.

Fig. 12
Fig. 12

Image illuminance (relative), field width, and resolution as a function of minimum N.A. γn. Calculations are for an objective with N.A. = 0.32. A, image illuminance for objective with specular reflection characteristics (calculation is for the center of the field); B, image illuminance for object with diffuse reflection characteristics; C, field width for a spherical surface with 7.8-mm radius (the cornea); D, resolution in lp/μm for lines perpendicular to the scan direction.

Equations (10)

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2 h = w / tan θ n .
u / r = γ n / γ x = ( n sin θ n ) / γ x ,
h < t ,
A = r 2 cos 1 ( u / r ) u 2 tan cos 1 ( u / r ) ,
A = r 2 cos 1 ( γ n / γ x ) ( γ n / γ x ) 2 tan cos 1 ( γ n / γ x ) .
I s = w s A ,
w s = M w ,
I d = w s A 2 ,
d = λ [ n 2 ( N.A. ) 2 ] 1 / 2 ( N.A. ) 2 ,
w = λ ( n 2 γ x 2 ) 1 / 2 γ x 2 tan sin 1 ( γ n n ) .

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