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Confocal microscopy using oblique sections for measurement of corneal epithelial thickness in conscious humans J. I. Prydal, M. G. Ken- Muir, F! N. Dilly, M. C. Corbett, S. Verma and J. Marshall

Department of Ophthalmology, Addenbrooke’s Hospital, Cambridge, UK

ABSTRACT. Purpose: Thickness measurements by confocal microscopy in conscious human subjects may be liable to error as a result of instability of the eye or instrument. Our aim was to evaluate a technique which was expected to be less sensitive to such problems. Mefhod. Thickness of corneal epithelium was determined from oblique confocal sections through cornea. A contact lens of known thickness worn by subjects was used to calibrate images. Results: There were two layers in images which could have corresponded to the stromal/epithelial interface. The mean result in each subject ranged from 38 to 53 pm using the more superficial layer and 46 to 60 pm using the deeper one. The smaller values gave the distance between the epithelial surface and the sub- epithelial nerve plexus and thus seemed to correspond to epithelial thickness. Conclusions: Measurements of epithelial thickness by our new method are com- parable with results of earlier studies.

Key words: confocal microscopy - corneal epithelium - epithelial thickness - cornea.

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onfocal microscopy gives images of C thin optical sections through the cornea at high resolution and contrast (Dilly 1988). It is a significant advance in the methods available for examining living tissue.

In earlier work, we used confocal microscopy to measure thickness of the tear film and epithelium in several animal species (Prydal & Campbell 1992). At that time clear images were only possible in completely immobile tissues and there- fore freshly sacrificed animals were ex- amined. Surfaces within the cornea were identified and the position of animals’ heads adjusted so that images were circu- lar. Optical sections were then tangential to surfaces, which could be accurately lo- cated. The separation between them, radial thickness, was measured using the stage micrometer of the microscope.

However, accurate measurements by

this method are not possible in conscious humans. Involuntary eye and head move- ments introduce unknown errors. An al- ternative method of measurement is pre- sented in this paper. Thickness of layers in the cornea are determined from single fields of video recordings with optical sections oblique to the eye surface. A soft contact lens of known thickness is worn by the subject and used for calibration. Optical sections pass through full thick- ness of contact lens and epithelium. The ratio of the separation of surfaces in im- ages is used to determine epithelial thick- ness. We have used this method before to measure the thickness of tear fluid be- tween contact lens and epithelium (Pry- dal & Dilly 1994). Here, measurements of epithelial thickness were made in the centre and at different positions across the cornea in healthy subjects.

Materials and Methods Experimental apparatus A Tandem scanning confocal microscope (Tandem Scanning Corporation, Virgi- nia, USA) was used with 100 W Mercury arc light source and x 24 water immer- sion objective (numerical aperture 0.6). The microscope was as described by Cavanagh et al. (1993), but was modified by replacing the base with a Zeiss table. Also, the observation optics were ad- justed to increase the field of view (630 by 420 pm). Optical sections are curved with the apex towards the objective and radius similar to that of the cornea (Per- sonal communication, J. Hill, Tandem Scanning Corporation). Cavanagh et al. (1993) determined the axial resolution of the microscope to be 9 pm by measuring the intensity of light reflected from a mir- ror.

A DAGE V T l O O O silicon intensified tube camera (SIT) was used and images recorded on S-VHS video tape with a Panasonic AG-7355 recorder. They were digitized at resolution 768 by 512 pixels and 256 gray levels (Imaging Technology Inc., Overlay Frame Grabber)

Contact lenses Contact lenses were specially manufac- tured by Contact Lens Precision Labora- tories (Cambridge, UK). Lenses were of hydroxymethylmethacrylate with 43% water content, refractive index 1.45 and

.diameter 14 mm. The central 12 mm was of constant radial thickness, nominally 60 pm. The manufacturing process was limited to tolerances of f 5%.

Thickness was measured after use. Lenses were cut normal to the surface, mounted between two saline soaked

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Stroma

sponges to maintain full hydration and the edge viewed using a standard micro- scope with calibrated eye-piece graticule, resolution 1.7 pm at x400 magnifica- tion.

Contact lenses were sterilized in 0.3% hydrogen peroxide.

Technique Images of oblique optical sections through the cornea were recorded on video. They showed the surface of the contact lens, the interface between con- tact lens and epithelium, and the surface of the stroma. The tip of the objective was 5 mm in diameter. Assuming the corneal radius of curvature to be 7.5 mm, the maximum angle of incidence possible without indentation of the cornea was about 40". Experiments were done with the objective at an angle of 20-30" to the normal at the eye surface.

Fig. l a is a scale diagram of an optical section through the eye surface. The opti- cal section is shown as flat and the contact lens and epithelium are assumed to be of the same refractive index and thickness. The ratio of the thickness of the contact lens in oblique sections (x', Fig. la) to that of the epithelium (y') was taken to be equal to the ratio of the radial thicknesses of the two layers (x/y):

x - x' Y Y' - _ - (see Fig. la.)

where x = radial thickness of contact lens y = radial thickness of epithelium

x' = apparent thickness of contact lens in optical section at oblique angle through the contact lens and epithe- lium

y' = apparent thickness of epithelium in same image.

Images were computer analysed to measure the distances x' and y'. Contact lens thickness, x, was measured by an in- dependent method, as described above. Epithelial thickness, y, was calculated from the equation.

Tests of accuracy Curvature of eye surface It was assumed in calculations that the contact lens and epithelium were flat par- allel layers. Errors introduced by the cur- vature of the eye surface were estimated using accurate drawings such as that in Fig. la. They were drawn at a scale of 300:l. The radius of the eye was taken to be 7.5 mm and the thickness of the epithe- lium and contact lens to be 50 pm each. Thus the stromal surface was of radius 225 cm, the outer surface of the epithe- lium 225.15 cm and external contact lens surface 225.30 cm. Optical sections were drawn for ten angles of incidence of the microscope -from 14" to 90". The ratio of the distance between intersections of the optical section with each surface (x'/y' in Fig. la) were measured from drawings. This was used to determine the error in the estimation of epithelial thickness at each angle. The additional error intro- duced by curvature of optical sections with a radius of 7.5 mm was estimated.

The procedure described above was re- peated drawing the optical sections as arcs with radii 225 cm.

Distances across images were cali- brated from images of a silicon crystal marked with squares of periodicity 9.9 pm (Agar Scientific Ltd, Stanstead, UK).

The approximate angle of optical sec- tions to eye surface in each image was determined from the thickness of the op- tical section through the lens (x', Fig. la) and its known radial thickness (x, Fig. la). Radius of curvature was taken as 7.5 mm. Those images in which the angle would have resulted in a significant error were excluded. The curvature of subjects' cor- neas was not measured. If it was as much as 1 mm different from the value used, the error in calculation of angle would have been less than 0.1".

Image distortion Confocal images were found to show pin- cushion distortion. It was quantified using the calibration silicon crystal.

Model of eye surface The method was tested by imaging cross sections through two soft contact lenses in contact with each other. They were supported on a hard contact lens. The ratio of the apparent thickness of the two lenses was determined from images. This was repeated after turning over the two soft lenses.

Procedure Four normal subjects, two male and two female, aged 32 to 72 years were exam- ined. The nature of experiments was ex-

Normal I

Direction of objective

I I J ' 30;'

Optical \-

Contact lens X I I\

I

Fig. la. Diagram of optics showing oblique optical section through con- tact lens, epithelium and superficial stroma. Objective at angle of 30" to normal. Optical section assumed to be flat, and contact lens and epithe- lium to be of same refractive index. Distances x'and y' determined from images and radial epithelial thickness, y, calculated from the ratio: x/y = x'/y' using known value of x, radial thickness of the contact lens.

1 7

I . I . I . I . 1 ' 1 ' 1 . 1 ' 1

1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

Angk to Normal

Fig. lb. Error in estimation of epithelial thickness resulting from as- sumption that contact lens and epithelium are flat parallel layers. The ratio x'/y' is plotted against the angle of the objective to normal. Radius of curvature of stromal surface taken to be 7.5 mm. Contact lens and epithelium each taken to be 50 pm thick and thus the ratio x'/y' would be 1.0 if the surfaces were flat.

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192 - a ? - E 3 120- - n

64-

plained fully and consent obtained. Eyes were anaesthetised with two drops of 0.4"/0 Oxybuprocaine Hydrochloride (Benoxinate) and the contact lens in- serted. The microscope was orientated at an angle of approximately 30" to the centre of the cornea and brought close to the eye. Methylcellulose (2O/n) was ap- plied to the objective before contact was made with the eye. Remote focusing con- trols were used to advance the optical section until images were cross sections through lens and epithelium. This was near to the end of its range, 1.0-1.5 mm from the objective tip.

Later, video recordings were reviewed and selected single fields digitized. A sample is shown in Fig. 2a. Surfaces of the contact lens, epithelium and stroma appeared as straight parallel bright bands. If these were not vertical, images were rotated a few degrees as this simpli- fied the image analysis process.

Images were brighter at the centre and dimmer towards the edge. This was a re- sult of uneven illumination and increased sensitivity towards the centre of the photocathode of the video camera. A flatter profile was obtained by subtract- ing a background image. This simplified further image analysis and enhanced the appearance of images. Ode background image was used for all subjects. It was made by advancing the plane of focus to image oblique sections through stroma. Fifty such images were digitized, aver- aged and then filtered until featureless and smooth in profile.

Five images from each subject were

analysed. The background was sub- tracted and, if necessary, brightness and contrast adjusted to identify structures. An area of approximately 5 12 by 256 pix- els was selected from as close as possible to the centre of images. Values in each column of the selected area were aver- aged and these results plotted. This showed peaks in brightness correspond- ing to the front surface of the contact lens ('a' Fig. 2b), the interface between con- tact lens and epithelium ('b' Fig. 2b) and the stroma ('d' Fig. 2b). A dark band at the stromal surface gave a trough in the curve ('c' Fig. 2 b). This could be clearly identified in all images. The co-ordinates of the highest point on each peak and the lowest point on the trough were deter- mined using a cursor. These values were chosen as the points corresponding to the interface between two layers as their loca- tion could be accurately determined in all images. Processing was done on a 486PC using the program Lucida (Kinetic Im- aging Ltd., Liverpool, UK).

Two values of epithelial thickness in images were determined from each image. One uscd the distance between the peak corresponding to the surfacc of the epithelium cb', Fig. 2b) and the peak from the stroma ('d', Fig. 2b). The second used the distance to the trough in the curve ('c', Fig. 2 b).

Attempts were made to correlate measurements of epithelial thickness with those made by scanning through the cornea with optical sections tangential to surfaces. This was done with the contact lens in the eye which was used to calibrate

axial displacement. The relationship was assumed to be linear within this range. Measurements were made in one subject who was particularly good at holding a stable head and eye position.

Oblique optical sections were also re- corded without a contact lens in the eye as subepithelial nerve plexuses could be precisely located.

Results Contact lens thicknesses One contact lens was used for all subjects. Thickness at 10 positions across the cen- tral 12 mm zone with parallel sides was 58.3 f 0.5 pm (mean f 95% confidence interval). A second lens used in tests of the method measured 51.2 k 3.9 pm.

Curvature of eye surface Fig. 1 b shows the error in measurement plotted against angle of incidence. At an angle of incidence of 20", the epithelial thickness would be overestimated by about 7%. At 30", the overestimate was 1%. All images were of angles between 20-30". The error that would have re- sulted from curvature of the optical sec- tion at a radius of 7.5 mm was not signifi- cant; it was too small to be detected in scale drawings. Results would have been more accurate had the microscope been orientated at larger angles of incidence. This was not possible as images were then less clear, probably because less light was reflected back to the objective at the in- creased angle to the reflecting surfaces.

I - I I I I S 1 ' 1

0 120 256 384 512 640 760

Horizontal position (pixels) Fig. 2a. Confocal optical section through contact lens, epithelium and superficial stroma. Single field of video recording which was digitized, the background subtracted and contrast enhanced. The section marked is that used for determining separation of surfaces. *a': Front surface of contact lens. %': Interface between contact lens and epithelium. 'c': Dark band at stromal surface. 'd': Bright band at stromal surface. ma1 surface.

Fig. 2b. Mean pixel intensity in each column of the image in Fig. 2a plotted against horizontal position across image. 'a': Front surfaceofcontact lens. 'b': Interface between contact lensand epithelium. 'c': Dark band at stromal surface. 'd': Bright band at stro-

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Image distortion Pin cushion distortion was restricted to the edge of images. Within the central re- gion of 640 by 384 pixels there was no significant distortion. At the edge of im- ages, the separation of two points was ap- proximately 2.5% greater than that in the centre.

Model of eye surface The ratio of apparent thicknesses in im- ages of cross sections through two con- tact lenses was used to test the technique. The measured ratio was 0.8 f 0.1 (actual ratio 0.9). After turning the two lenses over, the ratio from images was 1.1 f 0.03 (actual ratio 1.1). The interface between the two lenses was not clearly defined in images, presumably because they were of the same refractive index with little inter- veningfluid. Howevcr, the interface could be located with sufficient accuracy.

Epithelial thickness A sample image is shown in Fig. 2a and the mean pixel values in each column in Fig. 2 b.

A layer of tear fluid between contact lens and epithelium, 10-15 pm thick, was occasionally seen early in the examin- ation of a subject. It progressively re- duced in thickness ovef about one minute to less than could be resolved in confocal images: the reflection from the back sur- face of the contact lens and that from the surface of the epithelium merged. This is consistent with earlier work (Prydal & Dilly 1994). Measurements of epithelial

thickness were made only from those im- ages in which the lens appeared to be in contact with the epithelial surface. Thus the distance between peaks ‘a’ and ‘b’ (Fig. 2) corresponded to the thickness of the contact lens only - there was minimal if any fluid between the lens and epithelial surface.

Results in the four subjects are plotted in Fig. 3 a. Thickness measured from epi- thelial surface to the dark band (‘c’ Fig. 2b) ranged from 38.2 k 2.5 to 52.9 f 2.7 pm (mean f 95% confidence intervals). That measured to the peak (‘d’ Fig. 2b) ranged from 45.6 k 4.1 to 60.4 k 2.0 pm. The significance of these two measure- ments is reviewed below. Variation in thickness across the central 5 mm of the cornea is shown in Fig. 3 b. There was no significant difference in this area.

Epithelial thickness was also measured using axial scans through the cornea. Sur- faces gave bright reflections over a 15 pm change in position of the plane of focus. The range could be reduced by switching off automatic gain of the camera. Viewing images directly though the eye-piece re- vealed substantial chromatic aberration which was not evident on the mono- chrome video screen. This accounts for some of the inaccuracy when trying to locate a surface from video images alone. In addition, the mechanical support of the microscope was not stable. It could tilt by several degrees. The most superficial image showing bright reflections from a surface was taken as the location of the surface. The separation between epithe-

20

a 0

lial surface and the sub-epithelial nerve plexus was 39.3 pm and the depth to the first visible keratocytes 47.6 pm. Meas- urements by this technique were only possible in this subject because he was particularly good at maintaining stable head and eye positions. Thickness meas- ured using oblique optical sections in the same subject were 41.8 k 1.9 pm to the dark band at the stromal surface and 51.0f2.5 pm to the bright reflection from stroma.

Sub-epithelial nerves could occasion- ally be seen in oblique sections with a contact lens in the eye. They were seen more clearly in images of eyes without lenses. In both types of images nerves were located within the dark band, often close to the external surface of the bright reflection (Fig. 4).

Discussion In the earlier work of Calmettes et al. (19S6), Ehlers (1965) and Wolff (1968), measurements of epithelial thickness were made by examination of histological specimens. Results in man varied from 30 to 100 pm. Using high frequency ultra- sound, Reinstein et al. (1994) measured epithelium to be 41 to 54 pm in conscious humans subjects. Our measurements by confocal microscopy are comparable with these values. In earlier work we used confocal microscopy to measure epithe- lial thickness in nine animal species (Pry- dal & Campbell 1992). Values measured

-2.6 -1.15 0 +1.25 +2.5

Fig. 3a and b. a: Mean epithelial thickness in 4 subjects. Two values were calculated for each subject (see text). Error bars are + 95% confidence limits. b Epithelial thickness at 5 positions across the cornea in subject ALP. The smaller of the two values determined from images are plotted. Centre (0), two positions below centre (- 1.25 and - 2.5 mm) and two positions above centre (+ 1.25 and + 2.5 mm). Error bars are + 95% confidence limits.

Fig. 4. Confocal optical Section through epithelium and superficial stroma. There was no contact lens in the eye. Subepithelial nerves are within the dark band at the stromal surface. ‘b’: Interface between contact lens and epithelium. ‘c’: Dark band at stromal surface. ‘d’: Bright band at stromal surface.

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here in man were similar to that found in rabbit, rat, and two species of fish, but larger than measurements in gerbil, pi- geon, duck and two species of frog.

Ehlers (1965) reported the epithelium to be thicker at the edge of the cornea. Re- sults by confocal microscopy did not in- dicate any significant variation over the central 5 mm of the cornea. More periph- eral measurements were not made.

Wilson et al. (1980) measured epithe- lial thickness in human subjects using a modification of the pachymetry tech- nique developed by Maurice and Giardini (1951) for measurement of cor- neal thickness. It gave results from 55 to 67 pm. Values by confocal microscopy ranged from 38 to 60 pm, up to 29 pm smaller. The difference may perhaps be because measurements by pachymetry included the thickness of the pre-corneal tear film.

Oblique confocal optical sections through epithelium show a dark band superficial to a bright reflection from stroma. Attempts were made to identify the structure of these regions by scanning through the cornea with optical sections tangential to the eye surface. This method can give accurate results with stable im- mobile tissues and with direct visualiza- tion of images (Prydal & Campbell 1992). However, the technique is less accurate for examination of conscious human sub- jects because of involuntary head and eye movement. Also, the present equipment is not of sufficient mechanical stability and automatic gain of the video system makes localization of surfaces less pre- cise. Such errors are difficult to quantify. However, results of axial scans seemed to indicate that the dark band corresponded to the layer of dense sup-epithelial nerves and that the bright reflection was from superficial keratocytes within the stroma.

Images of eyes without contact lenses showed the subepithelial nerve plexus to be largely within the dark band at the stromal surface, in places close to the sur- face of the brighter reflection. Rozsa & Beuerman (1982) examined the neural structure of rabbit cornea. Dense plex- uses of nerves were found both deep and superficial to the basal lamina of epithe- lial cells. Thus this level, corresponding to the dark band in our images, may best define the interface between epithelium and stroma.

No correction was made for the dif- ference between the refractive index of epithelium and that of the contact lens used to calibrate images. Estimates of epithelial refractive index were reviewed by Clarke & Carney (1971). They varied

widely. Fischer (1928) used two different techniques and found values of about 1.54 and 1.41. Tagawa (1928) also found values of about 1.41 for rabbit corneal epithelium. Patel (1992) found similar values using an Abbe refractometer to measure refractive index in conscious human subjects, however, there may have been mucus and tear fluid between the in- strument and the epithelial surface. The contact lenses used in our experiments were of index 1.45, within the range found in these works. If the refractive index of epithelium was as low as 1.4, our results would be overestimates by less than 2 pm.

Measurement of thickness in con- scious subjects using oblique optical sec- tions through the cornea are probably more accurate than those using axial scans. Use of single fields of video recor- dings exclude inaccuracies from subject movement or instability of the instru- ment.

Acknowledgments We would like to thank the Ins Fund and Spe- cial Trustees of St. Thomas’ Hospital London for their generous support. M. C. Corbett holds the William’s Fellowship for Medical and Scientific Research.

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14: 105-120.

Received on November l l th, 1996.

Corresponding author: J. I. Prydal Department of Ophthalmology (Box 41) Addenbrooke’s Hospital Hills Road, Cambridge CB2 2QQ, UK.