Reports
out as the result of light returned in a combination of the dark-field and specular modes, but
the image of the slit suffers worse degradation
in this case as a result of interference from the
tissue on both sides (Fig. 1). The narrower the
slit the clearer the detail, and even in the case
of the endothelium the maximum useful width is
about 50 fim, which requires that a photographic
montage has to be constructed if an extended
view is to be obtained.
This disadvantage can be overcome if a narrow
slit is used and the specimen is slowly displaced
across it, while at the same time a film is moved
across a corresponding slit at the level of the
eyepiece. If t V rate and directions of movement
of the film correspond to the magnification of the
eyepiece, details of the image remain in register
on it during its exposure. A microscope has been
adapted for this purpose in which the stage is
shifted by means of a micrometer screw rotated
by a synchronous motor (Fig. 2). An identical
motor advances the 35 mm. film in the specially
constructed camera by means of rollers which
engage it along its edges. Pressure on the area
of the emulsion used to record the image was
found to produce artifacts. The microscope is
focused by means of observation through a hole
punched in the film, and the jaws of the camera
A scanning slit optical microscope. DAVID
M. MAURICE.
A transparent tissue is illuminated with a narrow slit of light which is formed by light transmitted down one-half of a microscope objective,
and the tissue is observed through the other half.
The tissue is moved slowly across the light, and
a photographic film is moved at a corresponding
rate over a parfocal slit at the eyepiece level. In
this way, thin optical sections are reproduced
on the film, free from light scattered in the bulk
of the tissue. Various comeal layers are illustrated,
and a system of alternating light and dark zones
in the stroma are revealed.
An adaptation of the optical microscope has
been previously described1 which allows the corneal endothelium of the intact eye to be examined
by means of the light reflected from its surface,
so-called specular microscopy. In this system,
light was passed down one-half of a 40x water
immersion objective and, after reflection, was observed down the other half. It was necessary
to use a relatively narrow slit of light focused
on the endothelial surface in order to avoid detail in the image being obscured by light scattered
from the overlying connective tissue, the stroma.
Structures within the stroma can also be made
Observation
Illumination
\ $mm>-J
\ksiiiMj
i
v
\
X
Fig. 1. Diagram illustrating the principle of the instrument. Illumination of the specimen
takes place through one-half of the objective and observation through the other, both fields
being defined by parfocal slits perpendicular to the plane of the figure. When the slit is
wide (right diagram), the details of the image are obscured by out-of-focus light scatter
from the surrounding tissue. When a narrow slit is used (left diagram), the scatter is very
much reduced, but the field of view is inconveniently small. A wider field is obtained by
slowly moving the observed object through the focal slit and recording the image on a film
which is undergoing a corresponding movement.
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lnoestigatioe Ophthdmdogy
1034 Reports
December 1974
Fig. 2. Diagram illustrating construction of the instrument. The specimen holder and the
film advance rollers were turned by identical synchronous motors. A hole could be punched
in the film through which the microscope was focused, and the jaws of the slit in the camera
could be adjusted to correspond to those in the illuminating system. The take-up chamber
can be detached and replaced so that film can be developed without having to readjust the
camera.
slit can be adjusted to correspond to those of
the illun~ination while focusing on a reflecting
surface under these conditions. Clutch mechanisms are provided for the rapid advance of the
film and for the readjustment of the stage
micrometer.
A variety of photographs of the corneal tissues
have been obtained in this way. The pictures
of the endothelium in the specular mode are
not only more extensive but improved in quality
over those with the stationary slit; the marks on
the surface do not appear to cast shadows but
are sharply delineated (Fig. 3). Descemet's membrane can be optically isolated, and the existence
of fibrous structures is revealed (Fig. 4 ) . In the
stroma the structure of the keratocytes can be
seen (Fig. 5), which corresponds to that seen
by staining." An unexpected discovery was the
appearance of alternnting bands of greater and
less scattering in the tissue which frequently
form a straight and regular pattern (Fig. 6 ) .
When viewed in the specular mode, the epithelial surface also shows a variety of features
that may be compared with its appearance in
scanning electron microscopy (Fig. 7).3
This system is easy to adjust and use with a
little experience. The illustrations were made with
a scanning slit 3 pm wide and a specimen advance rate of 127 pm per minute. Tri-X film
was used and was developed for 5 minutes in
Acufine. The Zeiss 40x water immersion objective
has a numerical aperture of 0.75, and geometry
indicates that the depth of the optical section
should be about equivalent to the slit width.
This has been checked by serial photographs
of a suspension of 1 pm diameter polystyrene
spheres embedded in gel as the microscope was
focused down in 1 pm steps. A sphere appeared
only in three successive photographs, in general.
It would be surprising if a similar instrument
had not been thought of previously, but I am
aware of no photographs of similar quality in
the literature. Another group has been working
on similar systems4~ using spot scanning through
the objective system. A close examination of the
principles on which they are constructed shows
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Volume 13
Number 12
Reports
m
Fig. 3. Specular reflection from surface of comeal endothelium of fresh isolated rabbit corneax450.
Fig. 4. Optical section through Descemet's membrane of fresh isolated rabbit cornea. The
presence of fibrous structures will be noted. Since no trace of the endothelium can be seen,
these structures must lie within the membrane or on its stromal surface. x450.
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Reports
Investigative Ophthalmology
December 1974
Fig. 5. Optical section through normal stroma of fresh isolated rabbit cornea. x450.
Fig. 6. Optical section through swollen stroma of fresh isolated rabbit cornea. x500.
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Volume 13
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Reports
1037
Fig. 7. Specular reflection from outer epithelial surface of fresh isolated rabbit cornea. x500.
that they would not evade the problem of scattering from neighboring tissue layers, as illustrated
in Fig. 1. Search of their cited references led
to a recent patent by Baer.': This instrument
operates on the principle of the present instrument and is superior in conception, since, if
enough light could be made available, it would
allow the optical section of the tissue to be
directly observed as well as photographed. No
photographs taken with this instrument have
been published to my knowledge, however. Goldmann7 made use of a similar technique to that
described here, at a different order of magnification, in his method of photographing the anterior
chamber.
I wish to thank Mr. Gunther Kuhn for engineering and constructing the modifications to the
microscope, and Miss Betty. Cassiman for taking
the photographs.
From the Division of Ophthalmology, Stanford
University Medical Center, Stanford, Calif. 94305.
This project was supported by National Institutes
of Health Grant EY-00431. Submitted for publication June 7, 1974.
Key words: scanning microscopy, corneal structure.
REFERENCES
1. Maurice, D. M.: Cellular membrane activity
in the corneal endothelium of the intact eye,
Experimentia 24: 1094, 1968.
2. Maurice, D. M.: The cornea and sclera, in:
The Eye, Davson, H., editor. Ed. 2. London,
1969, Academic Press, vol. 1.
3. Pfister, R. R.: The normal surface of corneal
epithelium: a scanning electron microscopic
study, INVEST. OPHTHALMOL. 12, 654,
1973.
4. Davidovits, P., and Egger, M. D.: Scanning
laser microscope for biological investigations,
Appl. Optics 10: 1615, 1971.
5. Petran, M., Hadravsky, M., Eager, M. D.,
et al.: Tandem-scanning reflected-light microscope, J. Opt. Soc. Am. 58: 661, 1968.
6. Baer, S. C : United States Patent 3, 547: 512.
7. Goldmann, H.: Spaltlampenphotographie und
-photometrie, Ophthalmologica 98: 257, 1940.
Histopathology of
tyrosine-fed rat.
keratopathy in the
E. BEARD,
MARGARET
ROBERT P. BURNS, LARRY F.
RICH, AND
EDWIN SQUIRES.
Young rats fed a diet containing excess l-tyrosine develop a reproducible and reliable keratopathy. This keratopathy clears spontaneously,
although the initiating dietary stitnidus is maintained. In less than 24 hours, edema of the central corneal epithelium develops, first in the basal
cells and then in focal full-thickness areas. These
areas of epithelial disease enlarge to form fullthickness "snowflake" opacities with cellular sepa~
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