International Ophthalmology 23: 199–206, 2001.J.R. Sampaolesi (ed.), Laser Scanning: Update 1, 17–24.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Confocal microscopy of the human cornea in vivo

Barry R. Masters & Matthias BöhnkeUniversitäts-Augenklinik, Inselspital, University of Bern, 3010 Bern, Switzerland

Key words: three-dimensional in vivo confocal microscopy, human cornea

Abstract

In vivo, scanning-slit, confocal microscopy offers improved resolution and has resulted in new discoveries ofcorneal pathology at the cellular level. The ability to provide high resolution, real-time images of the full thicknessof the living human cornea gives the clinician and the researcher an important new tool.

Introduction

The fundamental principles of noninvasive instru-mentation to investigate the eye are developed inseveral books [1–3]. There are classic works whichprovide the reader with an excellent introduction to thebiomicroscopy of the eye and the clinical use of theslit lamp [4–7]. Background information on confocalmicroscopy may be found in several sources [8–10].A recent reviews the historical development of opticaltechniques used for biomicroscopy of the eye, anddevelops the historical and theoretical foundations ofconfocal microscopy [11]. This paper critically dis-cusses the following topics: the practical techniquesof clinical confocal microscopy, the clinical findingsof normal, subclinical and pathological cases, the postsurgical cornea, and corneal preservation.

Biomicroscopy of the living eye from the slit lampto the confocal microscope

There is a direct and interesting lineage from theconfocal microscope developed by Goldmann, to thedevelopment of the specular microscope by Maurice,Koester and others, to the various types of clinical con-focal microscopes [11]. These instruments representpartial solutions to the problem of how to image thinoptical sections from a 500 micron thick, transparent,moving object-the human cornea in vivo.

The optical principle of the confocal microscopecan be shown in a schematic diagram (Figure 1). This

diagram shows a confocal microscope with two mi-croscope objectives; however for clinical in vivo use,a single objective lens is used to both illuminate anddetect the light scattered and reflected from the thickobject (cornea). A point source of light is focused toa point within the cornea by lens L1. The focusedlight illuminated a small volume at P1. A second lens,L2 collects the light from the same small volume P1and focuses the collected light from P1 at the slit S2,placed in front of the light detector. Both aperturesS1, and S2 are co-focused (“confocal”) on the samepoint in the focal plane that simultaneously is beingilluminated and detected. The confocal microscopediscriminates against light that is not in the focal plane.The out-of-focus light collected by lens L2 cannotenter the aperture at S2; thereby limiting the amountof out-of-focus light that is detected. This providesthe physical basis for the strong discrimination againstout-of-focus light in a confocal microscope. A con-focal microscope (two conjugate slits or apertures)provides increased lateral and depth resolution [8–10].A confocal microscope provides two enhancementscompared to a standard light microscope: enhancedlateral resolution and enhanced axial resolution. Thelatter property is the basis of its capability to opticallysection a thick, highly scattering specimen such as thecornea.

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Figure 1. The optical principle of a confocal microscope. S1 and S2 are confocal apertures. L1 and L2 are focusing lens for illumination anddetection respectively. P1 is the focal volume that is illuminated with the point source of light from S1 and focused at P1 with lens L1. P1 isimaged by lens L2 to form an image at the aperture S2. The light from focal volume P1 can enter the aperture S2. The out of focus light (dottedlines) only partially enters the aperture S2; thus, the depth discrimination.

Development of the scanning-slit confocalmicroscope

A book in which the history of confocal microscopy isdocumented as a series reprinted milestone papers andpatents from 1884 [Paul Nipkow patent] to the presentwas recently published [12]. This work shows the his-tory of the various types of confocal microscope andthe papers and patents that describe the developmentof the scanning-slit confocal microscope [13].

An alternative to point scanning uses a slit of il-lumination that is scanned over the back focal planeof the microscope objective [13]. This system hasthat advantage that many points are scanned in par-allel, markedly decreasing the scanning time. Anotherimportant advantage is the superior light throughputcompared with that in the point-scanning Nipkow disksystems. The disadvantage is that the microscope istruly confocal only in the axis perpendicular to the slitheight.

Thaer developed a scanning slit confocal micro-scope based on an oscillating double sided mirror(Figure 2). This design has the advantage of variableslit widths, a high numerical aperture objective, and a

halogen lamp as the light source [13]. Scanning anddescanning of the cornea is accomplished with an os-cillating two sided mirror. This design follows fromthe original microscope constructed over twenty yearsago by Svishchev in Moscow [14, 15].

The image of a slit is scanned over the back fo-cal plane of the microscope objective. The slit widthcan be varied in order to optimize the balance ofoptical-section thickness and image brightness. Theinstrument is based on the double-sided mirror whichis used for scanning and descanning. This confocal mi-croscope used a halogen lamp for illuminating the slit.The detector is a video camera that acquires imagesat video rates. This confocal microscope can imagebasal epithelial cells and the adjacent wing cells inthe living human cornea due to its high light through-put. The design consists of two adjustable slits placedin conjugate planes of the confocal microscope. Bothscanning of the illumination slit over the back focalplane of the microscope objective and descanning ofthe reflected light from the object is accomplished withan oscillating two-sided mirror.

For corneal imaging, a 40×, a 63× or a 100× highnumerical aperture, water immersion microscope ob-

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Figure 2. Schematic diagram illustrating the optical system of a clinical real-time scanning-slit confocal microscope. The light source is ahalogen lamp. S1 and S2 are confocal slits, and the L’s are lenses. Objective is a water immersion microscope objective. One half of theobjective is used for the illumination of the cornea; the other half of the objective is used to collect the light form the cornea. The illuminationlight path is shown by black dots, and the collection light path is shown in white. The M’s are fixed front-surface mirrors; M-M is an oscillatingdouble-sided mirror that is used for both scanning and descanning. The descanned image is detected by an intensified video camera.

jective (Zeiss, Germany) can be used. The resolutionof the different objectives and the thickness of the op-tical section to some extent are influenced by the lightlevels used and the geometry and the reflectivity of thestructures studied.

The following design parameters were incorpor-ated into the video-rate, scanning slit confocal micro-scope.

• Nonapplanating, high numerical aperture, wa-ter immersion microscope objectives, Zeiss 40×,Zeiss 63×, Zeiss 100× microscope objectives areused.

• A methylcellulose gel to optically couple the tipof the microscope objective to the cornea. Thereis no applanation or direct physical contact, whichdeforms the cornea, and introduces artificial foldsand ridges in the cornea, between the objective andthe surface of the cornea.

• One half of the numerical aperture is used forillumination, and one half of the numerical aper-ture is used for collection of the reflected andfluorescence light.

• Optical sectioning in the plane of the cornea isobtained with two sets of conjugate slits. The slitheights are variable and adjustable.

• An oscillating, two sided mirror (bilateral scan-ning) is used for scanning the image of the slit overthe back focal plane of the microscope objective,

and for descanning the reflected and back scatteredlight collected by the microscope objective fromthe focal plane in the specimen.

• The light source is a 12 volt halogen lamp.• The scanning is synchronized with the read-out

of an interline CCD camera in order that the fullvertical resolution of the intensified CCD cameracould be utilized.

What are the advantages of using a scanning slitconfocal microscope such as is described and demon-strated in this paper? Slit scanning confocal micro-scopes have a much higher light throughput than con-focal microscopes based on Nipkow disk. This has twoconsequences. First, the illumination incident on thepatient’s eye can be much less. This allows for a muchlonger duration of the use of the confocal microscopeon the patient’s eye without the severe patient discom-fort and high light intensity that is necessary with theuse of the confocal microscope based on the Nipkowdisk. Second, it is possible to image the low reflectinglayer of wing cells that are immediately adjacent to thebasal epithelial cells in the normal human cornea. Thislayer of wing cells has been imaged, at video-rates, assingle video frames without the need for any analog ordigital image processing using the video-rate scanningslit confocal microscope. No other video-rate confocalmicroscope has been able to image these wing cells inthe normal, in vivo human cornea. This new confocal

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instrument has unique advantages of other confocalsystems. The bright, high-contrast confocal images ofthe wing cells and the basal epithelial cells demon-strate its unique optical characteristics [16]. The lowreflectivity of the wing and basal cells in the normalhuman cornea present a low-contrast benchmark testspecimen for various types of confocal microscopy.The high rejection of stray light and the narrow depthof field coupled with the high numerical aperture mi-croscope objective (1.0 NA) results in the ability ofthe instrument to image these cell layers clearly inthe live normal human cornea. The clear advantageof slit scanning confocal microscopes for ophthalmicdiagnostics and basic eye research is best appreciatedwhen the basal epithelium in the anterior cornea isimaged. The video-rate scanning slit confocal micro-scope provides high-contrast, high resolution imagesof both the wing cell and basal epithelial cells in thenormal in vivo human eye.

In 1990, Masters suggested that an internal lenssystem would permit focusing at different depthswithin the cornea; however in order to keep light lossesto a minimum, and therefore maximize the light sens-itivity of the z-scanning instrument it was decided touse a z-scanning microscope objective [8]. This cap-ability of a z-scanning confocal has been implementedinto the clinical video-rate, slit-scanning confocal mi-croscope. In addition to microscopic pictures with alateral movement of the scanning slit, a confocal meas-urement of the tissue reflectivity can be performed byan automated z-scan through all layers of the cornea.For this procedure, the lateral scan is switched off.Reflectivity as recorded by a photomultiplier can beused to measure corneal haze, which may be of specialinterest in keratorefractive procedures.

Confocal images of the structures of the humancornea

The human cornea consists of the following layers:superficial epithelial cells, wing cells, basal epithelialcells, Bowman’s layer, stroma, Descemet’s mem-brane, and endothelial cells, from the tear film onthe anterior side of the cornea to the posterior side ofthe cornea adjacent to the aqueous humor [17]. Theunique optical properties of the cornea are consistentwith its morphology [16]. The normal cornea is avas-cular. At the anterior side is the corneal epithelium(about 50 microns thick at the central corneal region),under the epithelium is Bowman’s layer. It is 10–16

microns thick and is acellular except for the nerveswhich perforate it. It separates the epithelium from thestroma. The basal lamina of the epithelium is locatedon Bowman’s layer. The stromal region is about 450microns thick and contains large nerves, stromal kera-tocytes, and orthogonal layers of collagen fibers. Thearchitecture of human corneal nerves was studied withelectron microscopy [18, 19]. The morphology of thestromal keratocytes has been described with electronmicroscopy [20]. Posterior to the stroma is Descemet’smembrane, which is an acellular layer 15–20 micronsin thickness. The limiting layer on the posterior sideof the cornea is a single layer of corneal endothelialcells.

The standard histological sections, which give asagittal view of the corneal and, in this respect, arequite similar to the clinical biomicroscopy with the slitlamp. As the corneal layers and pathological findingsextend laterally, the clinician is used to move the slitlamp laterally as well, change the width and illumina-tion angle of the slit and thus collect three-dimensionalinformation about changes in tissue reflectivity andtheir location. In contrast, the confocal microscopecollects optical sections parallel to the corneal surface;all layers of the cornea can be imaged by changing thez-position of the objective. Consequently, the confocalflying slit microscope is an instrument which (i) allowsan immediate tangential view of the selected corneallayers under study and (ii) extends the diagnosticprinciple of biomicroscopy into the microscopic range.

An optical section of an in vivo human corneashows the intermediate cells of the anterior epithe-lium (Figure 3). An oblique optical section of thehuman cornea in vivo showing basal epithelial cellsand three nerves anterior to Bowman’s layer (Fig-ure 4). An interesting optical section of the corneashows a bifurcating cornea nerve in the anterior stroma(Figure 5). An confocal image of the in vivo humancornea at the level of the midstroma shows several ker-atocyte nuclei (Figure 6). In the lower left portion ofthe image (arrowhead) is a cell with several proceses.This may be a inflamatory cell in the storma. The en-dothelial mosaic of the central cornea can be observedwith the in vivo confocal microscope (Figure 7).

A more complete atlas of confocal microscopy ofthe normal cornea, in addition to typical images ofcorneal disease, corneal alterations induced by sur-gery, contact lens wear, and aging is available [11].In this extensive review the normal cornea is shown intwo sets of images; whole mount, fixed, stained lightmicroscopy and in vivo scanning slit confocal micro-

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Figure 3. (Sv37) A confocal optical section of the human cornea invivo showing the intermediate cells with their bright nuclei. The cellborders are highly reflective. The scale bar is 50 microns.

Figure 4. (BRM123) A slightly oblique confocal section of an invivo human cornea showing the polygonal cell borders of the basalepithelial cells on the left region of the image. The three bright linearstructures are nerves situated between Bowman’s layer and the basalepithelial cells. The scale bar is 50 microns.

scopy. This is another example of the importance ofcorrelative microscopy for the definitive identificationof ocular structures as observed in biomicroscopy.

The images obtained with the scanning-slit in vivoconfocal microscope are dependent on at least fourconsiderations: (1) the experience and skill of the ob-server, (2) the type of confocal microscope used, (3)the magnification and numerical aperture of the micro-scope objective, and (4) the type of image averagingand image processing and enhancement employed. Fi-nally, it has been our experience that even with a newly

Figure 5. (BRM176) A confocal optical section of an in vivo humancornea showing a nerve in the anterior stroma. The nerve bifurcatesand then the two sections recombine into a single nerve. The scalebar is 50 microns.

Figure 6. (BRM072) A confocal image of the in vivo human corneain the mid periphery of the stroma. The highly reflective nuclei ofseveral stromal keratocytes are shown with various shapes. In thelower left region (arrowhead) there is a cell with several processes.The scale bar is 50 microns.

installed commercial confocal microscope the imagequality of a confocal microscope is highly dependenton the optical alignment of the components.

Investigation of the cornea

Corneal alterations due to long term contact lenswear

This investigation shows how the use of a real-time,scanning-slit confocal microscope with a high numer-

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Figure 7. (M10) A confocal image of the in vivo human corneashowing the endothelial mosaic of the central corneal The highlyreflective dots are pigment particles. The scale bar is 50 microns.

ical aperture water immersion objective and singleframe review led to the discovery of a new cornealdegeneration [21]. The absence of frame averaging orother digital image processing was critical to the ob-servation, since frame averaging would preclude thedetection of the microdots that led to the discovery.A new type of chronic stromal aberrations has beenobserved in subjects with long-term contact lens wear.A real-time, scanning slit confocal microscope witha 50×/1.0 NA water immersion objective was usedto observe the cornea. The corneal optical sectionsfrom the superficial epithelium to the endotheliumwere recorded in real time without any further im-age processing and were reviewed frame by frame.This study confirmed the presence of epithelial mi-crocystic changes and alterations of endothelial cellmorphology which was previously described by oth-ers. The new and important finding of this study wasthe presence of highly reflective panstromal microdotdeposits in the corneal stroma. The dots were highlyreflective and had a size of 0.3 to 0.6 microns witha round-to-polygonal shape. The density and the sizeof the microdot deposits scales with the duration ofcontact lens wear. In patients wearing soft contactlenses for longer than 6 years, the microdots were ob-served all cased investigated. The microdots may belipofuscin or some other high reflective matter. Thisstromal microdot degeneration may be the early stateof a significant corneal disease, which eventually mayaffect large numbers of patients after decades of con-tact lens wear. A quantitative analysis of the densitydistribution of the microdots has been reported [22].

Corneal alterations following photorefractivekeratectomy (PRK)

A frequent and very legitimate question is what newclinical observations and discoveries have been dir-ectly linked to the use of the clinical confocal mi-croscope. The work of Bshnke et al. provides aninteresting illustration of how confocal microscopyled to the observation of persistent stromal changesafter myopic photorefractive keratectomy (PRK) inzero haze corneas [23]. In photo- refractive keratec-tomy treated patients and contact lens wearers, thebasal epithelial cells sporadically showed enhanced re-flectivity. However, rods and needles were observed inall photorefractive keratectomy treated patients, irre-spective of previous contact lens wear. In contact lenswearing controls, there were highly reflective granuleswhich were scattered throughout the thickness of thestroma; however, rods and needles were never ob-served [21]. The authors conclude that after 8 to 43months post photorefractive keratectomy there are ab-normal reflective bodies that persist beyond the timethat acute wound healing would be expected to becomplete. The clinical significance of these findingsin the context of visual acuity and long term status ofthe cornea is unknown.

A slit-scanning in vivo confocal microscope wasused to assess human corneal morphological charac-teristics after photorefractive keratectomy [24]. Eachlayer of the cornea was studied. The minimum follow-up time was 12 months. Fine linear structures wereobserved in the anterior stroma and in the midstroma,and a thin hyperreflective scar was noted after onemonth post PRK. These structures were more markedat 4 months, but were still present up to 26 months.The extension of these structures to the midstromaindicated that permanent corneal changes caused byPRK affect deeper stromal layers than the immediatesubepithelial region. Anterior stromal keratocyte dens-ity increased significantly 1 and 4 months after PRK.The midstromal and posterior keratocyte densities andendothelial cell densities were not affected. The sig-nificance of this investigation is that long-term altera-tions of the cornea in the midstroma could be observedover time with an in vivo confocal microscope.

Alterations of the human cornea during examinationwith an applanating confocal microscope

The use of confocal microscope for the examinationof the human eye in vivo is not without alterationsof the eye. The study by Auran et al. illustrates

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the flattening- induced effects of an applanating mi-croscope objective. They reported corneal bands andridges throughout the cornea following the contactwith an applanating microscope objective [25].

In addition to the previously discussed mechanicalflattening with the use of an applanating microscopeobjective there are several other sources of morpho-logical and physiological alteration with the use ofconfocal microscopy. First the use of anesthetic dropswhich contain preservatives effects the cell junctionsin the corneal epithelium. Second, the index matchinggel used between the tip of the microscope objectiveand the tear film of the cornea may dehydrate the sur-face cell layers of the cornea. Third, if microscopeobjective is not sterilized between patient examina-tions, there is a risk of bacterial and viral transmission.Finally, repeated daily examinations may lead to a lowgrade cellular reaction to the combined insults of anes-thetic drops and the index matching gel. We mentionthese possibilities as a matter of caution.

One way to mitigate these alterations of the corneais to use a noncontact confocal microscope [26]. Forexample, a long working distance air microscope ob-jective could be designed for in vivo observation of thecornea. The use of a noncontact microscope objectivehas many potential benefits for clinical observation ofpatients. There is not need for an index matching fluid;thus there is no physical contact with the ocular sur-face. There is no need for the use of anesthetic dropsin the patients eye. There is minimal chance of bac-terial and viral transmission from patient to patient.For these reasons we suggest and promote the devel-opment and use of noncontact confocal microscopy forthe observation of the living eye.

Three-dimensional confocal microscopy

Three-dimensional confocal microscopy provides aunique opportunity to visualize the in vivo cornea,ocular lens and optic nerve in three-dimensions [27–35]. It is the increases axial resolution of the confocalmicroscope that permits the sequential acquisition ofthin optical sections. The stack of optical sectionscan be converted to a three-dimensional volume in acomputer. It is then possible to observe the volumefrom any arbitrary angle and direction. It is the three-dimensional visualization of living cells and tissuesthat resulted in the ever increasing popularity of con-focal microscopy.

One problem that occurs in the acquisition of aseries of optical sections across the full thickness of

the human cornea in vivo is the lack of image re-gistration between sets of successive images. This isthe result of ocular motions due to rotations and themore significant problem of the motion of the globealong the optical axis due to the cardiac pulse. Oneapproach to this problem is to stabilize the globe witha low-vacuum suction cup system for stabilizing theeye in front of the microscope objective during thez-scan through the full thickness of the cornea [36].The authors had developed software for correction ofbackground illumination and small axial movements,and also calculated the keratocyte density.

Acknowledgements

The authors thank Dr. Andreas A. Thaer for his col-laboration in the development of the clinical confocalmicroscope, and Hans Kuenzli and his staff for theirexcellent work with the photographic prints.

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