Confocal microscopy of the eye

Abstract

Confocal microscopy of the living eye is becoming a valuable tool for diagnosis of retinal and corneal disease, as well as for monitoring and measuring corneal changes post-operatively. It is also useful for studying variations with time, such as nerve growth, keratocyte activation, and endothelial cell changes. The instrumentation is evolving, in response to challenges such as involuntary eye motion, the need for accurate depth and thickness measurements, and the desire for high quality images under a large range of specimen thicknesses, and variations in optical properties: index of refraction, absorption and light scattering.

Introduction

Confocal microscopy of the living eye is becoming a valuable tool for diagnosis, and for monitoring and measuring corneal changes post-operatively. It is also useful for studying variations with time, such as nerve growth, keratocyte activation, and endothelial cell changes. The instrumentation is evolving, in response to challenges such as eye motion, the need for accurate depth and thickness measurements, and the desire for high quality images under a large range of specimen thicknesses, and variations in optical properties: index of refraction, absorption, and light scattering.

The development and applications of confocal microscopy have been motivated by the ability of the instrument to perform optical sectioning. That is, to illuminate and image one layer of the specimen at a time, by excluding from the final image most of the light scattered or reflected from other layers. In the eye, it has proved useful in the cornea, conjunctiva, iris, lens, and retina, for its ability to visualize cells and subcellular details in tissue that produces significant backscattering of the incident light. The

thickness of the optical section, the layer that is free of light scattered or reflected from out-of-focus regions, is the measure of the effectiveness of the system.

The initial work in this area was done by Marvin Minsky,1 who wanted to see the interconnections between closely packed, interwoven cells in tissue of the central nervous system. The principle he employed is one that is still used today. In the illumination system of a light microscope, a pinhole is placed at a point that is confocal to the focal plane, so that a bright point of focused light is formed at the desired depth in the specimen. Light transmitted and scattered at this point is re-imaged at a second pinhole by the objective lens of the microscope. The two pinholes are ‘confocal’ because they share a common focal point.

Light that passes through the second pinhole is primarily light from the first pinhole that has travelled directly to its image in the specimen and then directly to the second pinhole, without significant scattering in the specimen. Light that is scattered from other points in the specimen will not be imaged at the second pinhole, so that little or none will pass through. This is the essence of optical sectioning: eliminating scattered light from planes other than the object plane. However, it is necessary to add a method of scanning in order to produce a two-di- mensional image. Minsky chose to use an x,y scan of the object, and to use a detector behind the second pinhole to generate a signal to the display. The first display was an available radar screen (early 1950s).

Minsky, who at the time was a medical student, described this configuration as “an elegant, symmetrical geometry: a pinhole and an objective lens on each side of the specimen”. He went on to design a system of confocal imaging for reflected light.

Fig. 1. The Tandem scanning microscope. Light from the source passes through a group of pinholes (indicated by the shaded area on the insert diagram), is transmitted through the beam-splitting mirror (oval), and is focused by the objective lens to the plane of focus. Light returning from the plane of focus is reflected from the beam-splitter mirror and forms an image at the under surface of the disc. Light that has been reflected or scattered from the plane of focus will form a pattern of bright spots that will be transmitted by the corresponding holes in the disc. As the disc rotates, the spiral array of pinholes will scan the full length and width of the image, which is then transmitted to the eye or camera. Light that is scattered from out-of-focus planes in the specimen will not be in focus when it reaches the disc on the return trip, and only a small fraction will pass through the pinhole array. (Reproduced from PetráÍ M et al.2 by courtesy of the publisher.)

The next major step was taken by PetráÍ and Hadravský2 when they incorporated a Nipkov disc3 in an incident light microscope (Fig. 1). This not only eliminated the need to physically scan the specimen, but it also produced an image that could be viewed directly by eye and recorded in color. With precision fabrication of the disc, the image can be effectively free of visible scan lines.

The system is quite effective because of the inverse square law, i.e., for a region of the object that is not exactly at the focal plane, light that passes through the image of a pinhole will be reduced in illuminance as the inverse square of the distance from that image. When the light is reflected or scattered back into the imaging system, the receiving pinhole will also be at a distance from the image of the out- of-focus specimen, and the light passing through the receiving pinhole will again be reduced by the inverse square law.

This design was adapted for the eye by the Tandem Scanning Corp., and more recently by Advanced Scanning, Ltd. Cavanagh, Jester, Petroll, and col-

Fig. 2. Corneal images from the Tandem scanning confocal microscope in the through-focus mode. A: Epithelial cells, corresponding to peak A in the graph. B: Basal-epithelial nerve plexus corresponding to peak B. C: Anterior layer of keratocyte nuclei (peak C). D: Stromal nerve corresponding to position D. E: Endothelial cell layer, corresponding to peak E. F: Three-dimensional reconstruction. G: Image intensity as a function of depth in the cornea. (Reproduced from Li et al.4 by courtesy of Swets & Zeitlinger)

leagues4,5 have developed further methods and instrumentation for quantifying image data and for improved imaging. Their through-focus imaging technique facilitates the recording of cells, nerves, haze, and pathology at all depths in the cornea (Fig. 2). As illustrated at G in Figure 2, a plot of the intensity variation during the scan provides a quantitative comparison of backscattered light at various depths.

An alternative to pinholes in a confocal system

is to illuminate and image through narrow slits. Gullstrand,6 in 1911, introduced slit illumination of the cornea. Oblique illumination with the narrow beam from the slit provided a form of optical sectioning as the thin sheet of light passed obliquely through the cornea. Under optimum conditions, it was possible to visualize endothelial cells because the oblique illumination separated the bright reflex of the anterior cornea surface from the much weaker image of the endothelial cell layer.

In 1968, David Maurice7 extended this principle to the microscope by imaging an illuminated slit onto the endothelial cell layer through one half of a microscope. The other half of the microscope was used to view the portion of the endothelial cell layer that was not obscured by the light reflected from the anterior cornea surface. First used in vitro, the principle later became widely used clinically as the specular microscope.

Not satisfied with the narrow image that could be obtained in this manner, in 1974 David Maurice8 developed a double slit scanning microscope for the in vitro examination of tissue. First the specimen was mounted on a moving stage, under an illuminated slit. Secondly, the light that was reflected specularly from the surface was imaged at the plane

of a stationary slit. Light passing through the second slit was focused on photographic film that was moving past the slit synchronously with the movement of the specimen. The jaws of the second slit served to block the reflex from the front surface of the cornea, but passed the light reflected from deeper layers. The specimen and the film were synchronously scanned so that the image of the specimen was laid down on the film continuously. The system produced excellent wide field images of glarefree images. This demonstrated that a double slit system could also be used for confocal imaging. It was not, however, suitable for in vivo studies.

Other confocal designs using slits were developed by Svishchev in 1969,9 Baer in 1970,10 and Brakenhoff and Visscher in 1992.11 None of these developments resulted in instrumentation for the eye, in vivo, but they contributed to the advancement of confocal microscopy in general.

The motivation for the slit scanning confocal microscope12 was to enable the specular microscope to become a wide field device (Fig. 3). As in the standard specular microscope, an illuminated (stationary) slit, S1 is imaged on the endothelial cell layer at the plane of focus, using slightly less than one half of the objective aperture. An oscillating mir-

Fig. 4. Stromal nerve junction, with filaments to keratocytes. Slit scanning confocal microscope, NA 0.8. (Photo by James Auran.)

ror, M2, is used to scan the illuminated slit image across a one-millimeter width of endothelium. Light reflected from this illuminated strip is directed through the other half of the microscope objective, to the second facet of the oscillating mirror. The second reflection reverses the action of the first reflection, stabilizing the light so that it can pass through the second slit, S2. The other function of the second slit is to block light that has been reflected or scattered from portions of the specimen that are not at the focal plane. Finally, the light that has passed through the second slit is sent to the third facet of the oscillating mirror M2. Reflection from this rotating facet causes the beam to scan across the image plane of the camera (film or CCD array) and to lay down the image strip by strip.

The thickness of the optical section is determined by the numerical aperture (NA) of the objective lens used, and the width of the confocal slits. When a very thin optical section is required, to visualize small details in the presence of scattered light from nearby structures, narrow slits will produce a thinner optical section, i.e., greater rejection of scattered light from adjacent layers.

However, if the desired details are weakly scattering and there is some degree of eye movement, wider slits will provide greater illumination, thereby minimizing the exposure time. Thus, the variable slit width can be utilized to allow the examiner to select the best section thickness, depending on the degree of detail in the image and the amount of eye movement of the subject.

Figure 4 is an image from the stroma of a normal subject. The nerve junction exhibits filaments extending to keratocytes. Figure 5 depicts another area where optical sectioning microscopy can be of value: the conjunctiva. Blood vessels, with associated nerves and white cells, are clearly imaged in spite of the density of the surrounding tissue and the close proximity of the sclera.13 Figure 6 illustrates the ante-

Fig. 5. Conjunctiva: blood vessels and associated white cells. Bar: 100 m. The slit scanning confocal microscope, NA 0.75. (Photo by Aryan Shayegani et al.13)

Fig. 6. Bovine lens, ex vivo. Anterior lens fiber cells. Slit scanning confocal image, NA 0.75. (Photo by Norman Kleiman.14)

rior region of a bovine lens, ex vivo utilizing the slit scanning confocal microscope, NA 0.80.14 Figure 7 is a human iris, in vivo, taken with the extended range objective, NA 0.80, on the slit scanning confocal microscope.

A widely used, important confocal system for the eye is the scanning laser ophthalmoscope, applied in the confocal mode.15 (See Fig. 8.) A focused, low power laser beam is reflected from a scanning mirror and projected into the eye and focused on the retina. On returning from the retina and emerging from the eye, it reflects again from the scanning mirror. This stabilizes the beam, so that it passes through a pinhole to a detector. The scanned image is then displayed on a monitor. The pinhole serves

Fig. 7. Human iris, in vivo. The pupillary margin, including the pigment ruff and anterior border layer, in a 37-year-old individual with a green iris. Slit scanning confocal microscope, NA 0.75. (Reproduced from Kummer et al.14 by courtesy of the publisher.)

Fig. 8. The scanning laser ophthalmoscope. Light from laser, L, passes through an acousto-optical modulator, AO, and a beam expander, B, then reflects from a multifaceted rotating mirror, H, which imparts a horizontal scan. The vertical scan is added by mirror, V. The laser beam is focused by the optics of the eye to the retina and is scanned over a small area. Light returning from the retina (dashed lines) follows the reverse path, bypasses the small mirror, T, passes through pinhole aperture, A, and is detected at D. The signal then goes to the display. The system is confocal because the laser beam behaves as if it had come from a point source. It can also be used in a non-confocal mode by removing the pinhole aperture, A. (Reproduced from Koester12 by courtesy of the publisher.)

Fig. 9. Human fundus, imaged by the scanning laser ophthalmoscope in the confocal mode. (Reproduced from Webb15 by courtesy of the publisher.)

Fig. 10. Schematic diagram of the optical system of the Confoscan3 confocal microscope.

to eliminate or greatly reduce the light that is scattered from regions in the eye other than at the focus of the laser beam. Figure 9 illustrates the human fundus as imaged in the confocal mode of the scanning laser ophthalmoscope. Because the numerical aperture of the system is limited by the eye’s pupil diameter (NA = 0.08 for a 3-mm pupil, or 0.22 for an 8-mm pupil), the resolution will not be as high as that of a high NA microscope objective that is used on the cornea, for example. Also, the optical section thickness will be greater than that from a high NA objective. Nevertheless, the confocal design improves the image when there is light scattering material in the paths of the incident and reflected light and delivers sharply defined images of retinal structures.

Masters and Bohnke16 utilized confocal microscopy to construct three-dimensional, high resolution images of the cornea, in vivo. They have also utilized confocal microscopy to demonstrate that long-term contact lens wear can produce degenerative microdot deposits in the corneal stroma.17

A confocal system based on synchronously scanned slits has been described by Wiegand et al.16 Figure 10 illustrates the principle, as adapted to the Nidek Confoscan microscope. In the microscope objective, the illumination and imaging light paths are separated, and as they pass into the cornea. By synchronizing the scanning of the slits and the

Fig. 11. Post-LASIK deep stromal nerve. Confoscan3. (Reproduced with permission.)

Fig. 12. LASIK interface (very low keratocyte density). Confoscan3. (Reproduced with permission of Nidek.)

video image cycle it was possible to eliminate effectively any blur due to eye movements. The Nidek ‘Confoscan3’ utilizes these principles.

The objective lens does not touch the cornea, but utilizes the optical immersion principle with gel and the tear layer. Image recording is continuous on a digital camera, with instant storage of several hundred frames. The width of the slits, together with the NA of the objective, determines the thickness of the optical section. It is estimated to be 5-10 m in the captured images.

Figure 11 shows a post-LASIK deep stromal nerve. Figure 12 shows a LASIK interface.

With the Confoscan3 system, the depth of any particular feature in the cornea can be deduced from a series of 350 images obtained in a fast scan through the cornea. If eye movement is detected, or suspected, the depth scan can be repeated.

Applications of confocal microscopy in refractive surgery

Specific applications of confocal microscopy in refractive surgery are listed and described in a comprehensive review paper by Ambrosio and Harrison.19 Wound healing has been observed and quantified to

determine thickness of the flap, total corneal thickness, and changes over time. Vesaluoma et al.20 documented flap alignment and followed the wound healing at the margin of the LASIK flap. The authors were also able to follow keratocyte myofibroblast transformation and activation, and proliferating fibroplastic cells. These wound healing events had been documented previously in primate models by Jester et al.21 Complications in wound healing with LASIK were studied with confocal microscopy by Vesaluoma et al.22

Post PRK wound healing and cornea nerve growth were studied by Linna et al.23 Complications following LASIK, e.g. particles at the interface, were documented by Kaufman et al.24

The high resolution and optical sectioning capabilities of confocal microscopy facilitate the study of keratocytes and their behavior following laser surgery. Patel et al.25 quantified the density of keratocytes as a function of depth in normal eyes, a statistically significant decrease with age, and a concentration of keratocytes in the anterior stroma in most but not all subjects. Moller-Pedersen et al.26 observed large numbers of wound healing keratocytes with higher reflectivity and haze development, all related to refractive instability.

Sub-epithelial and stromal nerves are particularly well imaged by high numerical aperture confocal microscopy. Linna et al.27 studied re-innervation after LASIK and correlated changes in neurotropic epitheliopathy with nerve regeneration into the flap. The growth pattern of basal epithelial nerves has been documented by Auran et al.28 Basal epithelial cell and unidentified cellular elements were also observed to move relative to established landmarks.

Other applications of confocal microscopy, including detection and diagnosis of corneal disease have been presented by Cavanagh et al.,29,30 by Auran et al.31 and by Florakis et al.32 Kleiman and Auran33 recently reported on the dynamics of Langerhans cells in traumatized cornea. The cells were identified by their morphology.

What future developments and refinements can be expected in confocal microscopy of the eye?

A number of improvements and new developments are possible. The list should include the following:

1. Objectives with higher numerical aperture, for better resolution, improved optical sectioning, and greater light gathering power, particularly for weakly scattering details.

Higher NA can improve both the optical sectioning and resolution of the system. One difficulty is that higher NA usually is accompanied by:

a.shorter working distance; and

b.smaller range of depth in the specimen within which the image quality is ‘diffraction limited’ (i.e., the image is essentially as perfect as diffraction and the wavelength of the light will permit).

The problem of smaller range of depth requires a change in the usual design of an objective lens. A well corrected objective designed for water immersion, NA 0.7, will give a diffraction limited image through the full 0.5-mm thickness of the cornea, but the same is not true for NA 0.8. (This problem is the same as that for cover glasses with high NA objectives.)

For examination of the lens, one problem is that the thickness of the lens is too great to obtain a good image at all depths using a standard design, high NA objective. In addition, there is variation in the depth of the anterior chamber between patients, so that an even greater range of focal depth is needed.

In order to utilize objectives with NA 0.8 and greater, a variable focus design can be utilized. The basic design is analogous to the familiar zoom lens system in that one optical element is moved axially relative to the other. But, with the zoom system, the magnification is changed while the image and object foci remain fixed. In the variable focus design, the position of the focal plane in the object is changed, while the image focal plane remains fixed. With the variable focus system, NA 0.8, the full thickness of the cornea can be examined without loss of resolution. And, for patients with various anterior chamber depths, the full thickness of the lens can be examined without loss of resolution.34 There is a small change in the magnification as the focus is changed, just as there is with a conventional lens used for the same purpose.

2. Improved (thinner) optical sectioning. Measured values of optical section thickness, provided by manufacturers.

The confocal slit, divided aperture systems for the eye have generally used equal, crescent shaped apertures for illumination and imaging (Figs. 3 and 10). For non-laser sources, e.g., arc lamps, this configuration provided the maximum efficiency for utilization of the available light.

However, when a laser can be used, the full power of the laser can be injected through a small portion of the objective aperture. When that portion is close to the edge of the objective aperture, two benefits are available. First, a larger portion of the objective aperture is then available for the imaging rays. This leads to better resolution in the image, as well as a brighter image and/or shorter exposure time. At the same time, the optical sectioning capability can be increased, because the angular spacing between the illumination and imaging ray bundles is greater (due to the small diameter of the laser beam). This improvement could also be feasible with the combination of a bright non-laser source and a very sensitive detector system.

The optical section thickness provided by each microscope needs to be well defined, so that comparisons can be made between various methods. With the confocal slit system there is a calculable depth (relative to the focal plane) beyond which no incident light can be scattered or reflected (by the specimen) into the imaging light beam. The calculation requires

knowledge of the locations of focal planes and the aperture stop of the objective. However, this calculation does not include the effects of stray light that may arise in the optics of the system due to reflections from lenses or scattering from surfaces.

With pinhole systems, a precise value for the optical section ‘thickness’ cannot be calculated. The intensity of the incident light decreases as the inverse square of the distance from the image of the pinhole, so the optical section does not have a well-defined thickness. For light scattered in the specimen, and travelling back through the objective, the fraction passing through the pinhole on the imaging side, the same inverse square law applies. If the optical section profile is to be calculated, the design of the optics needs to be known, and the precision of the pinhole locations and diameters are important.

Therefore, it would be useful for manufacturers to measure (and publish) the optical sectioning characteristics of the instruments. Not only is it more practical than calculating a theoretical value, but also it is more realistic, since the measurement will include light reflected and scattered at lens and other internal surfaces in such a way that a fraction of the light finds its way back to the image plane. The method for measurement is simple, in principle. A reflector, preferably diffusely reflective, is positioned at the focal plane, mounted on a platform that can be moved along the axis of the microscope.35 The light that returns to the focal plane is then measured as a function of the distance that the reflector is moved away from the focal plane. For immersed systems, the fluid layer must of course be maintained between the objective and the reflecting target.

This technique has also been useful in locating and identifying sources of stray light in the microscope, other than back scattering from the specimen. For example, when the measured light intensity no longer decreases as the distance to the target increases, it becomes apparent that there is stray light in the system coming from a source other that the reflecting surface.

3. Enriched image acquisition, in the presence of the inevitable eye movement.

Involuntary eye movements provide a major challenge for the user of a high magnification device such as the confocal microscope. Contacting the cornea with a polished glass surface similar to that of the applanation tonometer can help to stabilize the eye, but it does not prevent eye movement. For many patients, non-contact, using a gel or a fluid, would probably be their choice. In both cases the problems for the examiner are substantial. At high magnification, locating and then focusing on the detail of interest, the task is somewhere between challenging and impossible. One approach is to turn on the video camera or the recording CCD camera, and guide the microscope slowly and repeatedly through the volume of interest, to provide the opportunity to catch an image that is not blurred by motion. Reviewing stored CCD images or video tape in a search for usable images can be done at one’s leisure.

Fig. 13. The basal epithelial nerve plexus. The pachymeter reading of 0.040 mm represents the depth of the (in focus) nerves from the surface of the cornea. The depth measurement is derived from the focus setting of the objective lens. Slit scanning confocal microscope. (Photo by C. Koester.)

Digital cameras and image recording may offer another possibility. When the examination begins, the camera can be turned on so that each image is stored in temporary storage (T). When the examiner sees a good image, the SAVE button (or foot switch) is pressed. That causes the images that have been stored in the previous few seconds to be transferred to a longer term digital storage (L). As long as the SAVE button is being pressed the new images will be stored in (L) as well.

When the SAVE button is released, the new images are again sent to the temporary storage (T). When this temporary storage is filled, the next images can be written over the older images in (T), and the cycle can be repeated. Variations on this theme are also possible.

Other information could be added to the saved images: the examiner’s spoken comments at the time the SAVE button is activated, data available from the instrument such as focus setting, time and date, objective being used, patient ID, etc. The saved file would then be an enriched sample of images from the examination, not totally dependent on the reaction time of the examiner who is trying to catch the sharp image before the next eye movement. For both contact and non-contact instrumentation it will be helpful to have image recording systems that do not require reviewing the entire examination on tape (or on digital media) in order to find the acceptable images.

4.Improvement in the quality of acquired images by optimizing the alignment of the microscope objective to the cornea.

The instrument will, of course, record the optically sectioned image of whatever is at the focal plane. But the quality of that image will depend on the homogeneity, indices of refraction and thicknesses of the media between the objective tip and the focal plane, as well as any movement of the specimen that occurs during the exposure.

Contact with the cornea eliminates one source of image degradation: the astigmatism that is introduced when a diverging bundle of rays passes at an angle through a surface separating two media of different indices of refraction. When the microscope is used in the non-contact mode (with tear layer or gel), it is not easy to assure that the axis of the microscope is aligned perpendicular to the surface. For example, if the microscope axis was first aligned to the visual axis, then moved without rotation 2 mm to the right, the tilt of the cornea surface at that point would introduce a 50% increase in the blur circle diameter for a point located at the endothelial surface, and the image would no longer be diffraction limited. The magnitude of this effect increases with the NA of the objective. It also increases in proportion to the difference in index of refraction between the cornea and the medium separating the objective tip and the cornea.

Hence, one procedure is to be meticulous about aligning the microscope perpendicular to the cornea surface. Having the subject rotate the eye to the new area to be examined is not sufficient; the radius of the cornea is less than the distance from cornea to the center of rotation of the eye, so that the angle between the cornea surface and the front surface of the contact element increases as the eye is rotated.

Another procedure is to align (approximately) the microscope to the cornea by eye, and then applanate with the tip of the microscope to assure contact and therefore alignment. Another possible method is discussed in section 6, below.

5.Recording the location of the image.

The location of the image: the depth and to the

extent possible, the x,y position on the cornea is potentially important information: for returning to the same location at a later date, for reporting results, for documenting any change in thickness of the cornea, etc. In instruments using the contact mode, the depth information can be made available from the focus setting. An example of a pachymeter reading that is recorded on photographic film beside the cornea image is shown in Figure 13.

For systems used in the non-contact mode, a fast axial scan can locate the focus relative to two reference depths, e.g., the endothelial and epithelial surfaces. (See Petroll et al.4) Because of the frequency of eye movements, the measurement of depth should be based on two such scans. If the two fast scans show a difference in depth of the image, this may indicate that there has been eye motion between the two scans,

and the depth measurement should be flagged as being unreliable.

The measurement of x,y position is open to good ideas. An accurate determination of x,y location would be useful, but just the ability to return to the general area would be satisfactory in many cases. In one investigation, it was found that distinctive configurations of nerve junctions in the anterior cornea could be recognized and used to return to the same portion of the cornea a few days later.19 However, there were other, stationary landmarks in the cornea, and the nerve structures were observed to move during a period of several days relative to these stationary landmarks. Conclusion: Nerves in the anterior cornea, while quite visible and distinguishable, can be used as landmarks for relocating a position on the cornea for only a few days.

Stationary landmarks that were found useful in this study were: the corneoscleral limbus, anterior stromal nerves (including the subepithelial nerve plexus), entry points where nerves emerged from Bowman’s layer into the epithelium, stromal banding and basal lamina ridges induced by pressure from the flat contact element, and the nuclei of nonneural stromal cellular elements.28,36

An approximate position of the contact element on the cornea could be recorded by a simple, small photographic or digital camera mounted on the head rest. It could image the contact element, the lids and the visible portion of the limbus, which would probably be satisfactory for purposes of relocating the area of interest.

6.A gel that matches the index of refraction of the cornea (n = 1.376).

Such a gel would eliminate not only the astigmatism that is generated by a tilted cornea surface, but also the focusing power and spherical aberration caused by a spherical surface between two media with different refractive indices, including the cornea. If the dispersion (change of index with wavelength) also matched that of the cornea, chromatic aberration at that surface would also be eliminated. Of course, the gel should be viscous enough to stay in place on the cornea, but not so viscous that it would change the shape of the cornea during a movement of the eye or of the contact element of the objective lens. A gel used by Wiegand et al.18 has an index of 1.350, and is an improvement over the tear layer which has an index of about 1.34.

7.Image processing, particularly for motion blurred images.

Motion blurring in the plane of focus can now be corrected, post-exposure, by deconvolution methods. Deconvolution is a mathematical procedure that calculates the most likely object light distribution that would form the observed image, given the blur (the point spread function) produced by the lens when a point source is imaged. If the point spread function of the microscope is known, then deconvolution is

most effective. If not, a method called ‘maximum likelihood deblurring’ can be used.37

Confocal images generated by microscopes with thin optical sections should be excellent candidates for deconvolution, since they are relatively free from overlapping, out-of-focus images.

8.Reduction of stray light in the microscope. Confocal microscopy of nearly transparent tissue

is particularly susceptible to stray light. The source of the stray light is often illumination light that is reflected from lens surfaces, from beam splitter surfaces, or scattered from imperfections or dust on mirror and lens surfaces, or inner walls and lens mounts. If this reflected/scattered light cannot be prevented from reaching the image plane of the detector (film, CCD, or eye), the contrast in the image and the optical sectioning capability will be compromised.

The measurement of optical section thickness, as described above, is an effective technique for identifying sources of stray light. Another is to view the back aperture of the objective when the illumination light is on but there is no reflecting/scattering object at the focal plane. The back aperture of the objective should be appear black, as well as the rim of the aperture.

9. Confocal microscopy in other fields.

Confocal microscopy in other fields, particularly in other in vivo applications, can be a source of advanced techniques and technology. Corcuff et al.38 and Rajadhyaksha39 used the technology to study the skin. Khanna et al.40,41 combined laser heterodyne interferometry and confocal microscopy to measure vibration of hair cells and membranes in the cochlea of animals, in vivo.

10. Contact versus non-contact in confocal microscopy.

Normal eye motion is obviously a concern for high magnification by confocal microscopy. Contacting the cornea with a flat (or curved) end of the objective lens does not stop all eye motion, but it does establish the axial location of the epithelial surface. It also stops the motion of the cornea surface for periods of tenths of a second to several seconds, in most subjects.

With non-contact, the eye is free to move in any of its normal modes, which have been characterized by Charman42 as tremor, drift, and microsaccades and saccades. Tremors have high frequencies, but the amplitudes are small. Saccades have a velocity of about 700E/sec or more. This means that the corneal surface can move at a linear velocity of 153 mm/sec, or about 150 µm during a 0.001-second exposure. During the period between large eye movements, nearly continuous eye movement is observed in the eyepiece or viewfinder of the camera, which makes it difficult to select the detail to be photographed and to focus on it before it moves.

The use of objectives with higher NA can benefit the optical sectioning capability as well as resolution and image intensity.43 However, when the NA is in-

creased, the requirements on the rest of the optical system, including the eye being examined become more stringent. For example, an objective, NA 0.7, with gel or saline between the objective and the surface of the cornea can focus through the full thickness of the cornea and give diffraction-limited (optically perfect) images. However, if the NA is increased to 0.8, the performance begins to drop at a depth of about 0.4 mm. (This problem is the same as the cover glass thickness problem in laboratory microscopy.)

One way to overcome this obstacle is to use objectives that have been designed as variable focus lenses. These objectives have an internal moving component that allows the system to focus sharply over a range of thicknesses in the specimen. The first design for the eye allows the anterior surface of the lens to be examined in patients with anterior chamber depths from 2.4-4.0 mm, i.e., a range of variable focus of 1.6 mm, at NA 0.8.34

The above discussion assumes that the objective axis is aligned perpendicular to the surface of the - cornea, as it will be when the objective lens is in contact with the cornea surface. If non-contact microscopy is used, there are several potential optical problems, in addition to eye movement. As discussed in section 4, degradation of image quality occurs when the surface of the cornea is tilted relative to the axis of the microscope. Furthermore, the quality of the image will depend on the separation between the cornea and the tip of the microscope. One way to overcome this problem would be to develop a compatible gel whose index of refraction (and dispersion) match that of the cornea (n = 1.376). The objective lenses would also need to be designed for immersion in this medium.

11. Adaptive optics.

Pioneered by astronomers to improve the images of stars that are blurred by fluctuations in the atmosphere, adaptive optics are now being utilized to improve the resolution of retinal elements when observed through the subject’s cornea, aqueous, lens and vitreous.44 The aberrations of the eye’s optical system are first measured by sending a laser beam through the pupil, to the retina. The light returning through the pupil is then captured on an array detector. The data from this sensor are utilized to deform a mirror in such a way that the aberrations introduced by the eye can be significantly reduced. When the retina is to be photographed, the deformed mirror is incorporated into the imaging system, enabling a much improved image of the retina to be recorded.44

When the confocal microscope is used for examination of the cornea or lens, adaptive optics do not have the same advantages that they have in astronomy and retinal examination. Firstly, there is not a strongly reflective surface such as the retina that is close to the focal plane. Secondly, the surfaces that produce the aberrations (the corneal surfaces and lens) are relatively close to the detail that is to be photographed. Therefore, the correction that must be applied to the wavefront will depend on the location of the detail of

interest relative to the aberrating regions. In this case however, the aberrating surfaces are relatively close to the details of interest, so that their effect on resolution in the cornea and lens will be less than their effect on retinal imaging.

In summary, it appears to be likely that adaptive optics combined with high NA confocal microscopy will provide superb imaging of the retina. But the advantage of the combination for imaging the cornea and the lens is not so obvious. However, the image quality attainable with confocal imaging in the cornea and the lens is already excellent, and can become even better with objectives having higher NA, reduced stray light, and a greater range of focus.

Conclusions

Research studies using confocal microscopy have demonstrated that detailed images of normal and pathological corneas, lenses and retinas can be obtained. Changes over time, for example healing and nerve fiber growth, can be documented. Depths and thicknesses in the cornea can be measured.

For clinical applications some important instrument capabilities are: the efficiency and accuracy of obtaining sharp images, acquiring depth information, and having the ability to return to the same location. These functions need to be optimized for the clinical situation. Just as the slit lamp went through many refinements before reaching its present mature design, the confocal microscope may require additional effort on the part of practitioners, instrument designers, and manufacturers in order to realize a fully clinical, state- of-the-art ophthalmic instrument.

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Addendum

Status of instruments cited

•The Confoscan3 microscope is manufactured by Nidek.

•The scanning laser ophthalmoscope is manufactured by Heidelberg Instruments.

•The slit scanning confocal microscope is a research model at Columbia University, Department of Ophthalmology. The optical sectioning microscope is a research instrument at the Department of Otolaryngology, Columbia University.

•The Tandem scanning microscope is not presently available.