SPECULAR MICROSCOPY O F H U M A N CORNEAL E N D O T H E L I U M IN VIVO W I L L I A M M. BOURNE, M.D.,
AND HERBERT E. KAUFMAN,
M.D.
Gainesville, Florida 1
In 1968 Maurice introduced the specular microscope to observe endothelial cells in situ. Important modifications were made by Laing, Sandstrom, and Leibowitz2 to obtain photographs of endothelium in vivo. We made further changes so that the instrument can be easily used in the routine clinical examination and photography of the corneal endothelium at high magnification (200X). MATERIAL AND METHODS
The clinical specular microscope was used during patient examination (Figs. 1 and 2 ) . Light from a 6-V viewing lamp was passed through a slit aperture and condensing lens. The slit beam was directed by a system of mirrors out through the objective lens of the microscope and into the cornea. The objective was a 20 X water immersion lens with a flat-surfaced glass dipping cone ex tension that applanated the cornea, as in placing an applanation tonometer on the eye. The dipping cone may be adjusted by turn ing a knob to focus the image at different corneal thicknesses. The slit of light was re flected from the endothelium and returned back through the objective, with part of the light passing onward through the 10 X eyepiece and forming an image on the clear viewing screen of the single lens reflex camera. The Zeiss viewing lamp was focused in the same plane as a xenon flash tube, which allowed clear photographs to be taken (Kodak Tri-X film) despite continuous small eye movements. Topically applied anesthetic From the Department of Ophthalmology, Uni versity of Florida College of Medicine, Gainesville, Florida. This study was supported by Public Health Service grants EY 00033, EY 00446, and EY 00266 from the National Eye Institute, National Institutes of Health. Reprint requests to Herbert E. Kaufman, M.D., Department of Ophthalmology, University of Flor ida College of Medicine, P.O. Box J284, Gaines ville, FL 32610.
was applied to the cornea before the ex amination. The examination was no more uncomfortable for the patient than applana tion tonometry. The linear magnification of the image on film was approximately 70 X. The optical principles of the clinical specular microscope have been described elsewhere. The instrument was reliable and durable. Forty normal eyes of 40 white patients, 6 to 87 years of age, were examined with the specular microscope, and photographs of the central endothelium were obtained. All eyes were normal by slit-lamp examination and had no history of ocular abnormalities. Eyes with high refractive errors or increased in traocular pressure were excluded. The cen tral corneal thickness was measured in every eye by the same observer using a modified Haag-Streit pachometer.3 An estimation of endothelial cell density was made for each eye by counting the num ber of cells in a photographic field of known area. The area was calculated first by using the specular microscope to photograph the surface of a glass slide with calibration lines etched on it at 10-μ intervals. The photograph was taken through an aqueous medium with the dipping cone at the same distance from
Fig. 1 (Bourne and Kaufman). The clinical specular microscope in use. 319
320
AMERICAN JOURNAL OF OPHTHALMOLOGY
MARCH, 1976
Fig. 2 (Bourne and Kaufman). Schema of the clinical specular microscope with 1, viewing lamp; 2, condensing lens; 3, xenon flash tube ; 4, slit aperture ; S, condensing lens ; 6, objective lens ; 7, dipping cone lens; 8, attachment for focus ing ; 9, eyepiece lens ; and 10, view ing screen.
the objective lens as it would be when a cor nea of normal thickness is examined and photographed. This calibrated picture was then projected on a screen at a final central magnification of 400X (the 10-μ marks were 4.0 mm apart) and the screen was marked at each projected 10-μ interval. The endothelial photographs were then projected on the same screen at exactly the same magnifi cation and position, and the area of cells to be counted was calculated. We positioned the endothelial picture exactly as the calibration slide had been, since the peripheral magnifi cation was somewhat more than the central (the marks were 4.5 mm apart). The num ber of cells in three different photographs were counted and the results averaged. The area counted in each photograph was ap
proximately 0.04 mm2, and the readings were expressed as cells per square millimeter. RESULTS
Although small eye movements were pres ent during the examination, a clear view of the endothelium was obtained in all cases, and the details of cell size and pattern were discerned and photographs were of good quality. Each examination lasted only a few minutes and was not uncomfortable for the patient. The eyes were again examined with the slit lamp after specular microscopy, and we saw occasional tiny punctate central epi thelial irregularities that are often present after applanation tonometry. These disap peared within several hours and were not symptomatic.
Fig. 3 (Bourne and Kaufman). Normal endothelium of a 12-year-old boy. Cell density is 4,003 cells/ mm2. Bar gauge = 100 μ (0.1 mm) (X320).
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321
Fig. 4 (Bourne and Kaufman). Normal endothelium of a 74-year-old man. Cell density is 1,944- cells/ mm2. Bar gauge = 100 μ (0.1 mm) (X320). The endothelial mosaic was continuous and without detectable abnormalities in all 40 eyes. There was a difference in cell density between two patients, which represented the two extremes of mean cell size in this sam pling of the normal population (Figs. 3 and 4). A variability in individual cell size within each cornea was also present. The mean endothelial cell densities of the
40 normal patients were plotted as a function of patient age (Fig. S). A Student's f-test used to test the correlation coefficient be tween age and cell count showed that endo thelial cell density decreased significantly with increasing age ( P < .001). As a mea sure of reproducibility, the variability was calculated for each individual and was 161.4 cells/mm2 (one standard deviation).
CELLS/mm2 4000-1
3500-
3000-
2500-
2000-
1500-
'T IS
I
30
45
60
75
90
AGE (YEARS)
Fig. 5 (Bourne and Kaufman). Endothelial cell density of 40 normal eyes vs. age. A statistically significant linear regression of cell count with age is present. The sloping line represents a least squares regression of age vs. cell counts. The fitted linear model is Y = S8.49-0.138X, where Y represents the square root of the cell count and X represents age.
AMERICAN JOURNAL OF OPHTHALMOLOGY
322
MARCH, 1976
CELLS/mm2 4000-1
3500-
3000-
2500-
2000-
I500H
1
1
1
1
1
1
r-
.46 .47 .48 .49 .50 .51 .52 .53 CORNEAL
"i—i—i—i—i—i—r~ .54 .55 .56 .57 .58 .59 .60
THICKNESS (mm)
Fig. 6 (Bourne and Kaufman). Endothelial cell density vs. central corneal thick ness. No correlation is demonstrated.
The endothelial cell density vs. corneal thickness was plotted (Fig. 6). There was no correlation using the two sample f-tests between any of these three factors ( P > .05). DISCUSSION
This clinical specular microscope is ex tremely useful for studying and documenting the condition of the corneal endothelium at any time, such as before an intraocular surgi cal procedure, and for assessing the effects of aging, drugs, operations, inflammation, and the like, on the endothelial cells. In addition, it may be possible to demonstrate endothelial disease or damage before it is detectable by other methods of examination. We documented a gradual decrease in endo thelial cell density with age. Such a decrease was suggested in the past,4'5 but it could not be sufficiently documented. One study of
corneas from cadavers did not demonstrate a decrease with age.6 Bron and Brown7 studied endothelial cell density by using a corneal macrocamera, but their series of eight normal eyes was too small to demonstrate an age-related effect. This gradual decrease in the density of central endothelial cells repre sents a net loss of cells with time, indicating an apparent inability of these cells, under normal conditions, adequately to replace themselves by cell division. There was a lack of correlation between central endothelial cell density and corneal thickness. According to Ota, there is a direct relationship between endothelial permeability and central corneal thickness (unpunished data). Therefore, the observed loss of endo thelial cells with age does not normally affect the barrier function of the endothelium, which remains intact as long as there is enough spreading potential or "healing re-
VOL. 81, NO. 3
CORNEAL ENDOTHELIUM IN VIVO
serve." Endothelial decompensation with corneal edema occurs only beyond this point. In addition, we observed and photographed only the central cornea (approximately 4 mm in diameter) and no conclusions should be drawn regarding the peripheral endothelium.
SUMMARY
A clinical specular microscope used in the routine examination of the corneal endothe lium of 40 patients, at high magnification, without inconvenience or discomfort to the patients, detected endothelial damage or dis ease that was not seen by slit-lamp examina tion. A statistically significant decrease ( P < .001) in the number of central endothelial cells with age was documented.
323
REFERENCES
1. Maurice, D. M.: Cellular membrane activity in the corneal endothelium of the intact eye. Experientia 24:1094, 1968. 2. Laing, R. A., Sandstrom, M. M., and Leibowitz, H. M. : In vivo photomicrography of the corneal endothelium. Arch. Ophthalmol. 93:143, 1975. 3. Mishima, S., and Hedbys, B. O. : Measure ment of corneal thickness with the Haag-Streit pachometer. Arch. Ophthalmol. 80:710, 1968. 4. Kaufman, H. E., Capella, J. A., and Robbins, J. E. : The human corneal endothelium. Am. J. Ophthalmol. 61:83S, 1966. 5. Stocker, F. W.: The Endothelium of the Cornea and Its Clinical Implications, 2nd ed. Springfield, Charles C Thomas, 1971, pp. 13-16. 6. Irvine, A. R., and Irvine, A. R., Jr.: Varia tions in normal human corneal endothelium. Am. J. Ophthalmol. 36:1,279, 1953. 7. Bron, A. J., and Brown, N. A. P. : The endo thelium of the corneal graft. Presented at the Ophthalmology Society of the United Kingdom meeting, April 1974.
OPHTHALMIC MINIATURE
The note from Scotland Yard enclosed an optician's specification identical with that sent by Mr. Crimplesham, and added that it was an unusual one, owing to the peculiar strength of the lenses and the marked difference between the sight of the two eyes. Dorothy L. Sayers Whose Body? 1923
AND HERBERT E. KAUFMAN,
M.D.
Gainesville, Florida 1
In 1968 Maurice introduced the specular microscope to observe endothelial cells in situ. Important modifications were made by Laing, Sandstrom, and Leibowitz2 to obtain photographs of endothelium in vivo. We made further changes so that the instrument can be easily used in the routine clinical examination and photography of the corneal endothelium at high magnification (200X). MATERIAL AND METHODS
The clinical specular microscope was used during patient examination (Figs. 1 and 2 ) . Light from a 6-V viewing lamp was passed through a slit aperture and condensing lens. The slit beam was directed by a system of mirrors out through the objective lens of the microscope and into the cornea. The objective was a 20 X water immersion lens with a flat-surfaced glass dipping cone ex tension that applanated the cornea, as in placing an applanation tonometer on the eye. The dipping cone may be adjusted by turn ing a knob to focus the image at different corneal thicknesses. The slit of light was re flected from the endothelium and returned back through the objective, with part of the light passing onward through the 10 X eyepiece and forming an image on the clear viewing screen of the single lens reflex camera. The Zeiss viewing lamp was focused in the same plane as a xenon flash tube, which allowed clear photographs to be taken (Kodak Tri-X film) despite continuous small eye movements. Topically applied anesthetic From the Department of Ophthalmology, Uni versity of Florida College of Medicine, Gainesville, Florida. This study was supported by Public Health Service grants EY 00033, EY 00446, and EY 00266 from the National Eye Institute, National Institutes of Health. Reprint requests to Herbert E. Kaufman, M.D., Department of Ophthalmology, University of Flor ida College of Medicine, P.O. Box J284, Gaines ville, FL 32610.
was applied to the cornea before the ex amination. The examination was no more uncomfortable for the patient than applana tion tonometry. The linear magnification of the image on film was approximately 70 X. The optical principles of the clinical specular microscope have been described elsewhere. The instrument was reliable and durable. Forty normal eyes of 40 white patients, 6 to 87 years of age, were examined with the specular microscope, and photographs of the central endothelium were obtained. All eyes were normal by slit-lamp examination and had no history of ocular abnormalities. Eyes with high refractive errors or increased in traocular pressure were excluded. The cen tral corneal thickness was measured in every eye by the same observer using a modified Haag-Streit pachometer.3 An estimation of endothelial cell density was made for each eye by counting the num ber of cells in a photographic field of known area. The area was calculated first by using the specular microscope to photograph the surface of a glass slide with calibration lines etched on it at 10-μ intervals. The photograph was taken through an aqueous medium with the dipping cone at the same distance from
Fig. 1 (Bourne and Kaufman). The clinical specular microscope in use. 319
320
AMERICAN JOURNAL OF OPHTHALMOLOGY
MARCH, 1976
Fig. 2 (Bourne and Kaufman). Schema of the clinical specular microscope with 1, viewing lamp; 2, condensing lens; 3, xenon flash tube ; 4, slit aperture ; S, condensing lens ; 6, objective lens ; 7, dipping cone lens; 8, attachment for focus ing ; 9, eyepiece lens ; and 10, view ing screen.
the objective lens as it would be when a cor nea of normal thickness is examined and photographed. This calibrated picture was then projected on a screen at a final central magnification of 400X (the 10-μ marks were 4.0 mm apart) and the screen was marked at each projected 10-μ interval. The endothelial photographs were then projected on the same screen at exactly the same magnifi cation and position, and the area of cells to be counted was calculated. We positioned the endothelial picture exactly as the calibration slide had been, since the peripheral magnifi cation was somewhat more than the central (the marks were 4.5 mm apart). The num ber of cells in three different photographs were counted and the results averaged. The area counted in each photograph was ap
proximately 0.04 mm2, and the readings were expressed as cells per square millimeter. RESULTS
Although small eye movements were pres ent during the examination, a clear view of the endothelium was obtained in all cases, and the details of cell size and pattern were discerned and photographs were of good quality. Each examination lasted only a few minutes and was not uncomfortable for the patient. The eyes were again examined with the slit lamp after specular microscopy, and we saw occasional tiny punctate central epi thelial irregularities that are often present after applanation tonometry. These disap peared within several hours and were not symptomatic.
Fig. 3 (Bourne and Kaufman). Normal endothelium of a 12-year-old boy. Cell density is 4,003 cells/ mm2. Bar gauge = 100 μ (0.1 mm) (X320).
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CORNEAL ENDOTHELIUM IN VIVO
321
Fig. 4 (Bourne and Kaufman). Normal endothelium of a 74-year-old man. Cell density is 1,944- cells/ mm2. Bar gauge = 100 μ (0.1 mm) (X320). The endothelial mosaic was continuous and without detectable abnormalities in all 40 eyes. There was a difference in cell density between two patients, which represented the two extremes of mean cell size in this sam pling of the normal population (Figs. 3 and 4). A variability in individual cell size within each cornea was also present. The mean endothelial cell densities of the
40 normal patients were plotted as a function of patient age (Fig. S). A Student's f-test used to test the correlation coefficient be tween age and cell count showed that endo thelial cell density decreased significantly with increasing age ( P < .001). As a mea sure of reproducibility, the variability was calculated for each individual and was 161.4 cells/mm2 (one standard deviation).
CELLS/mm2 4000-1
3500-
3000-
2500-
2000-
1500-
'T IS
I
30
45
60
75
90
AGE (YEARS)
Fig. 5 (Bourne and Kaufman). Endothelial cell density of 40 normal eyes vs. age. A statistically significant linear regression of cell count with age is present. The sloping line represents a least squares regression of age vs. cell counts. The fitted linear model is Y = S8.49-0.138X, where Y represents the square root of the cell count and X represents age.
AMERICAN JOURNAL OF OPHTHALMOLOGY
322
MARCH, 1976
CELLS/mm2 4000-1
3500-
3000-
2500-
2000-
I500H
1
1
1
1
1
1
r-
.46 .47 .48 .49 .50 .51 .52 .53 CORNEAL
"i—i—i—i—i—i—r~ .54 .55 .56 .57 .58 .59 .60
THICKNESS (mm)
Fig. 6 (Bourne and Kaufman). Endothelial cell density vs. central corneal thick ness. No correlation is demonstrated.
The endothelial cell density vs. corneal thickness was plotted (Fig. 6). There was no correlation using the two sample f-tests between any of these three factors ( P > .05). DISCUSSION
This clinical specular microscope is ex tremely useful for studying and documenting the condition of the corneal endothelium at any time, such as before an intraocular surgi cal procedure, and for assessing the effects of aging, drugs, operations, inflammation, and the like, on the endothelial cells. In addition, it may be possible to demonstrate endothelial disease or damage before it is detectable by other methods of examination. We documented a gradual decrease in endo thelial cell density with age. Such a decrease was suggested in the past,4'5 but it could not be sufficiently documented. One study of
corneas from cadavers did not demonstrate a decrease with age.6 Bron and Brown7 studied endothelial cell density by using a corneal macrocamera, but their series of eight normal eyes was too small to demonstrate an age-related effect. This gradual decrease in the density of central endothelial cells repre sents a net loss of cells with time, indicating an apparent inability of these cells, under normal conditions, adequately to replace themselves by cell division. There was a lack of correlation between central endothelial cell density and corneal thickness. According to Ota, there is a direct relationship between endothelial permeability and central corneal thickness (unpunished data). Therefore, the observed loss of endo thelial cells with age does not normally affect the barrier function of the endothelium, which remains intact as long as there is enough spreading potential or "healing re-
VOL. 81, NO. 3
CORNEAL ENDOTHELIUM IN VIVO
serve." Endothelial decompensation with corneal edema occurs only beyond this point. In addition, we observed and photographed only the central cornea (approximately 4 mm in diameter) and no conclusions should be drawn regarding the peripheral endothelium.
SUMMARY
A clinical specular microscope used in the routine examination of the corneal endothe lium of 40 patients, at high magnification, without inconvenience or discomfort to the patients, detected endothelial damage or dis ease that was not seen by slit-lamp examina tion. A statistically significant decrease ( P < .001) in the number of central endothelial cells with age was documented.
323
REFERENCES
1. Maurice, D. M.: Cellular membrane activity in the corneal endothelium of the intact eye. Experientia 24:1094, 1968. 2. Laing, R. A., Sandstrom, M. M., and Leibowitz, H. M. : In vivo photomicrography of the corneal endothelium. Arch. Ophthalmol. 93:143, 1975. 3. Mishima, S., and Hedbys, B. O. : Measure ment of corneal thickness with the Haag-Streit pachometer. Arch. Ophthalmol. 80:710, 1968. 4. Kaufman, H. E., Capella, J. A., and Robbins, J. E. : The human corneal endothelium. Am. J. Ophthalmol. 61:83S, 1966. 5. Stocker, F. W.: The Endothelium of the Cornea and Its Clinical Implications, 2nd ed. Springfield, Charles C Thomas, 1971, pp. 13-16. 6. Irvine, A. R., and Irvine, A. R., Jr.: Varia tions in normal human corneal endothelium. Am. J. Ophthalmol. 36:1,279, 1953. 7. Bron, A. J., and Brown, N. A. P. : The endo thelium of the corneal graft. Presented at the Ophthalmology Society of the United Kingdom meeting, April 1974.
OPHTHALMIC MINIATURE
The note from Scotland Yard enclosed an optician's specification identical with that sent by Mr. Crimplesham, and added that it was an unusual one, owing to the peculiar strength of the lenses and the marked difference between the sight of the two eyes. Dorothy L. Sayers Whose Body? 1923
