In Vivo Photomicrography of the Corneal Endothelium Ronald A.
Laing, PhD;
Marita M.
Sandstrom; Howard M. Leibowitz, MD
technique and apparatus for observand photographing the corneal endothelium in vivo at a magnification of approximately \m=x\200 is described. The method is suitable for animal experimentation and for diagnostic observation and clinical research in humans. A
ing
Maurice1
described 1968, Inspecular microscope
photographing
a new
suitable for the corneal endothe¬
lium in situ. A subsequent report sug¬ gested the use of this instrument for examining the corneal endothelial layer of enucleated, intact human do¬ nor eyes prior to keratoplasty.2 Modi¬ fications of the instrument and the technique have been used to show morphological changes in the corneal endothelium of the enucleated rabbit eye resulting from the presence of air in the anterior chamber3 and from storage at 4 C (our personal observa¬
tions, 1973).
This communication reports an advance in instrumentation and tech¬ nique that makes possible photo¬ micrography of the corneal endothe¬ lium in vivo. The method is applicable to both animal and human subjects and allows examination of changes in cellular structure as a function of time at a magnification of approxiSubmitted for publication Sept 26, 1973. From the Ophthalmic Biomedical Engineering Laboratory and the Massachusetts Lions Eye Research Laboratory, Department of Ophthalmology, Boston University School of Medicine, Boston. Reprint requests to Boston University School of Medicine, 80 E Concord St, Boston, MA 02118
(Dr. Laing).
mately 200 to living subject.
be
performed
on
the
Materials and Methods The Maurice specular microscope was removed from its supporting stand and the rack-and-pinion travel mechanism at¬ tached to a specially designed horizontal mount that coupled the microscope ocular to a photomicrographic camera via a shock-absorbing structure. For in vivo endothelial photography, the apparatus was mounted on a slit-lamp biomicroscopic base (Zeiss). The joystick and height adjust¬ ment of the base and the original focusing adjustments of the microscope allowed the system to be aligned relative to the sub¬ ject's eye. Coarse focus was accomplished by rotating the ring on the objective lens. Fine focus was provided by a rack-andpinion focus adjustment of the photomicro¬ graphy camera mount. The original lamp and lamp housing were replaced by a high intensity quartz iodide lamp powered by a voltage supply that provided a switch allowing the lamp voltage to be rapidly changed from 6 direct current (used for viewing the endo¬ thelium) to 12 direct current (used for photographing the endothelium). The orig¬ inal microscope objective was replaced with a x20 water-immersion objective that was coupled to a contact lens (Lo-Vac) for human endothelial photography. Use of this arrangement incorporating a suctiontype contact lens greatly reduced the
contact with the epithelial surface in a manner similar to that described by Mau¬
original work on endothelial photography of the enucleated eye.1 This procedure produced sufficient ocular rice in his
stabilization in the anesthetized animal so that respiratory movements did not affect the sharpness of the image at an exposure time of one fifteenth second. Cats, rabbits, and monkeys were photographed by this method; no special adaptation was needed for each species other than the means to position the head so that the rabbit eye was stabilized to the side and the cat and monkey eye anteriorly. Photographs of humans were taken af¬ ter application of a topical anesthetic agent. The subject was seated with his head stabilized in the conventional head¬ rest used for slit-lamp biomicroscopy. The instrument was aligned to the subject's eye, the contact lens affixed, coarse focus adjusted, fine focus adjusted, lamp voltage increased momentarily to 12 and photo¬ graphs taken on 35-mm film (Kodak TriX) using an exposure time of one-fifteenth second. During the procedure, the subject experienced no discomfort. Instillation of fluorescein into the cul-de-sac following the procedure showed only mild surface rough¬ ening of the corneal epithelium similar to that seen after tonometry. Figure 3 shows the procedure applied to the human sub¬ ject. The linear configuration of the photo¬ graphs results from the use of slit-lamp il¬ lumination to show the endothelium.
amount of eye motion encountered and per¬
mitted photomicrographs to be taken with¬ out retrobulbar anesthesia. The instru¬ mentation is diagrammed in Fig 1 and 2.
Animal photographs were taken on anesthetized subjects without the contact lens. The water-immersion lens was posi¬ tioned on the optic axis of the cornea in
Results
The method described allows micro¬ structure of the corneal endothelium of the living eye at magnifications of up to x200 and permits photographic recording
scopic observation of the
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Fig 1 —Modification of Maurice specular reflection microscope for in vivo endothe¬ lial photomicrography. A, Film holder. B, Shock-absorbing mount. C, Viewing op¬ tics. D, Light source. E, Ocular coupling. F, Specular microscope. G, Water-immer¬ sion objective with suction contact lens. H, Slit-lamp biomicroscope mount. I, Rack-and-pinion focus apparatus. Fig 2.—Objective used for human in vivo endothelial photomicrography. A, Waterimmersion objective (Nikon) with dipping cone
(original magnification
20).
,
Suction contact lens with central opening to accommodate A. C, Coarse focus ad¬
justing ring.
A turgescence.4 Damage to this cellular
layer interferes with its physiological function with resultant edema, de¬
Fig
3.—Procedure for human in vivo endothelial
Fig 4.—Rabbit
endothelium in vivo
photomicrography.
(original magnification
100).
image. Figure 4 shows the ap¬ pearance of the rabbit endothelium in vivo, Fig 5 that of the cat, and Fig 6 that of the monkey. The photomicrographic appearance of the living human endothelium is shown in Fig 7. of the
Comment
Fig 5.—Cat endothelium magnification 100).
in vivo
(original
A normally functioning corneal en¬ dothelium is necessary for corneal de-
creased corneal transparency, and vi¬ sual loss. Histopathological studies, using fixed, stained flat preparations of the cornea, have shown morpholog¬ ical changes in the cells of the endo¬ thelium associated with age,5 inflam¬ mation,6 and ocular disease.71" Information correlating these morpho¬ logical changes with abnormal physi¬ ological function in vivo is limited. The apparatus described in the present study permits direct micro¬ scopic visualization of the corneal en¬ dothelium in vivo and has provisions for recording the image photograph¬ ically. It is suitable for animal experi¬ mentation and for diagnostic obser¬ vation and clinical research in humans. The photographs of animals presented were made with the sub¬ jects under general anesthesia. A suc¬ tion-type contact lens constructed specifically for the species under study and local infiltration type of an¬ esthesia and akinesia could possibly permit experimental study in animals without the need for general anesthe¬ sia. The methods for human in vivo endothelial photomicrography require only a topical anesthetic and modi¬ fication of the suction contact lens in general clinical use. Since they pro-
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/22/2015
This study was supported in part by Public Health Service grants EY0054 and EY00042 from the National Eye Institute, by a grant from the Massachusetts Lions Eye Research Fund, Inc., Boston, and by a grant from Research to Prevent Blindness, Inc.
References
endothelium in vivo
(original magnification
100).
Fig 7.—Human endothelium in vivo (original magnification
100).
Fig 6.—Monkey
vide for the noninvasive observation and study of the endothelial cells in the living subject, documentation of morphological changes can be made over a period of time, whether these changes represent the normal aging
process, the results of disease or sur¬ gical trauma, or the response to spe¬ cific controlled experimental stimuli. Dynamic correlations can be made be¬
morphological alterations and changes in physiological function. tween
1. Maurice DM: Cellular membrane activities in the corneal endothelium of the intact eye. Experientia 24:1094, 1968. 2. Hoefle FB, Maurice DM, Sibley RC: Human corneal donor material: A method of examination before keratoplasty. Arch Ophthalmol 84:741-744, 1970. 3. Leibowitz HM, Laing RA, Sandstrom MM: Effect of air in the anterior chamber on the corneal endothelium. Arch Ophthalmol 92:227-230, 1974. 4. Mishima S, Kudo T: In-vitro incubation of rabbit cornea. Invest Ophthalmol 6:329-339,1967. 5. Oh JO: Changes with age in the corneal endothelium of normal rabbits. Acta Ophthalmol 41:568-573, 1963. 6. Smolin G: Corneal endothelial changes during the hypersensitivity reaction. Am J Ophthalmol 65:349-352, 1968. 7. Irvine AR, Irvine AR Jr: Variations in normal human corneal endothelium. Am J Ophthalmol 36:1279-1285, 1953. 8. Kaufman HE, Robbins JE, Capella JA: The endothelium in normal and abnormal corneas. Trans Am Acad Ophthalmol Otolaryngol 69:931\x=req-\ 942, 1965. 9. Polack FM: The effect of ocular inflammation on corneal grafts. Am J Ophthalmol 60:259\x=req-\ 269, 1965. 10. Wilcox WC, Wood EM, Oh JO, et al: Morphological and functional changes in corneal endothelium caused by the toxic effects of influenza and Newcastle disease virus. Br J Exp Pathol 39:601-609, 1958.
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/22/2015
Laing, PhD;
Marita M.
Sandstrom; Howard M. Leibowitz, MD
technique and apparatus for observand photographing the corneal endothelium in vivo at a magnification of approximately \m=x\200 is described. The method is suitable for animal experimentation and for diagnostic observation and clinical research in humans. A
ing
Maurice1
described 1968, Inspecular microscope
photographing
a new
suitable for the corneal endothe¬
lium in situ. A subsequent report sug¬ gested the use of this instrument for examining the corneal endothelial layer of enucleated, intact human do¬ nor eyes prior to keratoplasty.2 Modi¬ fications of the instrument and the technique have been used to show morphological changes in the corneal endothelium of the enucleated rabbit eye resulting from the presence of air in the anterior chamber3 and from storage at 4 C (our personal observa¬
tions, 1973).
This communication reports an advance in instrumentation and tech¬ nique that makes possible photo¬ micrography of the corneal endothe¬ lium in vivo. The method is applicable to both animal and human subjects and allows examination of changes in cellular structure as a function of time at a magnification of approxiSubmitted for publication Sept 26, 1973. From the Ophthalmic Biomedical Engineering Laboratory and the Massachusetts Lions Eye Research Laboratory, Department of Ophthalmology, Boston University School of Medicine, Boston. Reprint requests to Boston University School of Medicine, 80 E Concord St, Boston, MA 02118
(Dr. Laing).
mately 200 to living subject.
be
performed
on
the
Materials and Methods The Maurice specular microscope was removed from its supporting stand and the rack-and-pinion travel mechanism at¬ tached to a specially designed horizontal mount that coupled the microscope ocular to a photomicrographic camera via a shock-absorbing structure. For in vivo endothelial photography, the apparatus was mounted on a slit-lamp biomicroscopic base (Zeiss). The joystick and height adjust¬ ment of the base and the original focusing adjustments of the microscope allowed the system to be aligned relative to the sub¬ ject's eye. Coarse focus was accomplished by rotating the ring on the objective lens. Fine focus was provided by a rack-andpinion focus adjustment of the photomicro¬ graphy camera mount. The original lamp and lamp housing were replaced by a high intensity quartz iodide lamp powered by a voltage supply that provided a switch allowing the lamp voltage to be rapidly changed from 6 direct current (used for viewing the endo¬ thelium) to 12 direct current (used for photographing the endothelium). The orig¬ inal microscope objective was replaced with a x20 water-immersion objective that was coupled to a contact lens (Lo-Vac) for human endothelial photography. Use of this arrangement incorporating a suctiontype contact lens greatly reduced the
contact with the epithelial surface in a manner similar to that described by Mau¬
original work on endothelial photography of the enucleated eye.1 This procedure produced sufficient ocular rice in his
stabilization in the anesthetized animal so that respiratory movements did not affect the sharpness of the image at an exposure time of one fifteenth second. Cats, rabbits, and monkeys were photographed by this method; no special adaptation was needed for each species other than the means to position the head so that the rabbit eye was stabilized to the side and the cat and monkey eye anteriorly. Photographs of humans were taken af¬ ter application of a topical anesthetic agent. The subject was seated with his head stabilized in the conventional head¬ rest used for slit-lamp biomicroscopy. The instrument was aligned to the subject's eye, the contact lens affixed, coarse focus adjusted, fine focus adjusted, lamp voltage increased momentarily to 12 and photo¬ graphs taken on 35-mm film (Kodak TriX) using an exposure time of one-fifteenth second. During the procedure, the subject experienced no discomfort. Instillation of fluorescein into the cul-de-sac following the procedure showed only mild surface rough¬ ening of the corneal epithelium similar to that seen after tonometry. Figure 3 shows the procedure applied to the human sub¬ ject. The linear configuration of the photo¬ graphs results from the use of slit-lamp il¬ lumination to show the endothelium.
amount of eye motion encountered and per¬
mitted photomicrographs to be taken with¬ out retrobulbar anesthesia. The instru¬ mentation is diagrammed in Fig 1 and 2.
Animal photographs were taken on anesthetized subjects without the contact lens. The water-immersion lens was posi¬ tioned on the optic axis of the cornea in
Results
The method described allows micro¬ structure of the corneal endothelium of the living eye at magnifications of up to x200 and permits photographic recording
scopic observation of the
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/22/2015
Fig 1 —Modification of Maurice specular reflection microscope for in vivo endothe¬ lial photomicrography. A, Film holder. B, Shock-absorbing mount. C, Viewing op¬ tics. D, Light source. E, Ocular coupling. F, Specular microscope. G, Water-immer¬ sion objective with suction contact lens. H, Slit-lamp biomicroscope mount. I, Rack-and-pinion focus apparatus. Fig 2.—Objective used for human in vivo endothelial photomicrography. A, Waterimmersion objective (Nikon) with dipping cone
(original magnification
20).
,
Suction contact lens with central opening to accommodate A. C, Coarse focus ad¬
justing ring.
A turgescence.4 Damage to this cellular
layer interferes with its physiological function with resultant edema, de¬
Fig
3.—Procedure for human in vivo endothelial
Fig 4.—Rabbit
endothelium in vivo
photomicrography.
(original magnification
100).
image. Figure 4 shows the ap¬ pearance of the rabbit endothelium in vivo, Fig 5 that of the cat, and Fig 6 that of the monkey. The photomicrographic appearance of the living human endothelium is shown in Fig 7. of the
Comment
Fig 5.—Cat endothelium magnification 100).
in vivo
(original
A normally functioning corneal en¬ dothelium is necessary for corneal de-
creased corneal transparency, and vi¬ sual loss. Histopathological studies, using fixed, stained flat preparations of the cornea, have shown morpholog¬ ical changes in the cells of the endo¬ thelium associated with age,5 inflam¬ mation,6 and ocular disease.71" Information correlating these morpho¬ logical changes with abnormal physi¬ ological function in vivo is limited. The apparatus described in the present study permits direct micro¬ scopic visualization of the corneal en¬ dothelium in vivo and has provisions for recording the image photograph¬ ically. It is suitable for animal experi¬ mentation and for diagnostic obser¬ vation and clinical research in humans. The photographs of animals presented were made with the sub¬ jects under general anesthesia. A suc¬ tion-type contact lens constructed specifically for the species under study and local infiltration type of an¬ esthesia and akinesia could possibly permit experimental study in animals without the need for general anesthe¬ sia. The methods for human in vivo endothelial photomicrography require only a topical anesthetic and modi¬ fication of the suction contact lens in general clinical use. Since they pro-
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/22/2015
This study was supported in part by Public Health Service grants EY0054 and EY00042 from the National Eye Institute, by a grant from the Massachusetts Lions Eye Research Fund, Inc., Boston, and by a grant from Research to Prevent Blindness, Inc.
References
endothelium in vivo
(original magnification
100).
Fig 7.—Human endothelium in vivo (original magnification
100).
Fig 6.—Monkey
vide for the noninvasive observation and study of the endothelial cells in the living subject, documentation of morphological changes can be made over a period of time, whether these changes represent the normal aging
process, the results of disease or sur¬ gical trauma, or the response to spe¬ cific controlled experimental stimuli. Dynamic correlations can be made be¬
morphological alterations and changes in physiological function. tween
1. Maurice DM: Cellular membrane activities in the corneal endothelium of the intact eye. Experientia 24:1094, 1968. 2. Hoefle FB, Maurice DM, Sibley RC: Human corneal donor material: A method of examination before keratoplasty. Arch Ophthalmol 84:741-744, 1970. 3. Leibowitz HM, Laing RA, Sandstrom MM: Effect of air in the anterior chamber on the corneal endothelium. Arch Ophthalmol 92:227-230, 1974. 4. Mishima S, Kudo T: In-vitro incubation of rabbit cornea. Invest Ophthalmol 6:329-339,1967. 5. Oh JO: Changes with age in the corneal endothelium of normal rabbits. Acta Ophthalmol 41:568-573, 1963. 6. Smolin G: Corneal endothelial changes during the hypersensitivity reaction. Am J Ophthalmol 65:349-352, 1968. 7. Irvine AR, Irvine AR Jr: Variations in normal human corneal endothelium. Am J Ophthalmol 36:1279-1285, 1953. 8. Kaufman HE, Robbins JE, Capella JA: The endothelium in normal and abnormal corneas. Trans Am Acad Ophthalmol Otolaryngol 69:931\x=req-\ 942, 1965. 9. Polack FM: The effect of ocular inflammation on corneal grafts. Am J Ophthalmol 60:259\x=req-\ 269, 1965. 10. Wilcox WC, Wood EM, Oh JO, et al: Morphological and functional changes in corneal endothelium caused by the toxic effects of influenza and Newcastle disease virus. Br J Exp Pathol 39:601-609, 1958.
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/22/2015
