in vivo confocal microscopy of the bulbar conjunctiva

Original Article

In vivo confocal microscopy of the bulbarconjunctivaNathan Efron PhD DSc,1,2 Munira Al-Dossari MAppSc1,2 and Nicola Pritchard BAppSc(Optom)1,2

1Institute of Health and Biomedical Innovation, and 2School of Optometry, Queensland University of Technology, Kelvin Grove,Queensland, Australia

ABSTRACT

Background: The aim of this work is to develop amore complete qualitative and quantitative under-standing of the in vivo histology of the human bulbarconjunctiva.

Methods: Laser scanning confocal microscopy(LSCM) was used to observe and measure morpho-logical characteristics of the bulbar conjunctiva of 11healthy human volunteer subjects.

Results: The superficial epithelial layer of the bulbarconjunctiva is seen as a mass of small cell nuclei. Cellborders are sometimes visible. The light grey bordersof basal epithelial cells are clearly visible, but nucleican not be seen. The conjunctival stroma is com-prised of a dense meshwork of white fibres, throughwhich traverse blood vessels containing cellularelements. Orifices at the epithelial surface may rep-resent goblet cells that have opened and expelledtheir contents. Goblet cells are also observed in thedeeper epithelial layers, as well as conjunctivalmicrocysts and mature forms of Langerhans cells.The bulbar conjunctiva has a mean thickness of32.9 � 1.1 mm, and a superficial and basal epithelialcell density of 2212 � 782 and 2368 � 741 cells/mm2, respectively. Overall goblet and matureLangerhans cell densities are 111 � 58 and 23 � 25cells/mm2, respectively.

Conclusions: LSCM is a powerful technique forstudying the human bulbar conjunctiva in vivo andquantifying key aspects of cell morphology. Theobservations presented here may serve as a useful

marker against which changes in conjunctival mor-phology due to disease, surgery, drug therapy orcontact lens wear can be assessed.

Key words: conjunctiva, goblet cell density, Langerhanscell density, laser scanning confocal microscopy._ 335..344

INTRODUCTION

The conjunctiva is a thin, translucent mucous mem-brane that extends from the eyelid margin to thecorneal limbus. Thus, it forms a large sac-like struc-ture that prevents extraneous matter from passing bythe side of the globe into the orbit.1 Mucus secretedby the conjunctiva forms an integral part of the pre-ocular tear film that in turn maintains a moist, hydro-philic ocular surface.1

In a clinical environment it is possible to examinethe conjunctival surface at up to 40¥ magnificationusing a slit-lamp biomiscoscope.2 Changes in thelevel of vascular engorgement of the conjunctiva canbe assessed, and gross tissue irregularities andabnormalities can be detected. Vital dyes such asfluorescein, rose bengal and lissamine green can beused to reveal conjunctival surface disorders suchas abnormal wetting or interruptions to tissueintegrity.3

Until recently, our understanding of the micro-scopic anatomy of the conjunctiva has been derivedfrom light and electron microscopic studies ofexcised tissue samples.1 Additional informationrelating to the surface anatomy of the conjunctiva hasbeen gathered using the technique of impressioncytology,4 whereby superficial conjunctival cells areremoved from the ocular surface by briefly pressinga small cellulose acetate or absorbent paper strip

� Correspondence: Professor Nathan Efron, Institute of Health and Biomedical Innovation, and School of Optometry, Queensland University of Tech-

nology, 60 Musk Avenue, Kelvin Grove, Qld 4059, Australia. Email: [email protected]

Received 20 October 20008; accepted 2 April 2009.

Clinical and Experimental Ophthalmology 2009; 37: 335–344 doi: 10.1111/j.1442-9071.2009.02065.x

© 2009 The AuthorsJournal compilation © 2009 Royal Australian and New Zealand College of Ophthalmologists

mailto:[email protected]

against the conjunctiva of a living subject. Surfacecells that remain adhered to the paper are examinedmicroscopically with the assistance of stainingagents. This technique has been particularly usefulin estimating the goblet cell density (GCD) of theconjunctival surface in disease5 and in response todrug therapy,6 ocular surgery7 and contact lens wear.8

When white light tandem- and slit-scanning con-focal microscopes were introduced towards the endof the 20th century, it became possible to investigatetransparent structures, such as the cornea, at a cellu-lar level in vivo at up to 500¥ magnification.9 With themore recent introduction of the in vivo laser scanningconfocal microscope (LSCM), it has become possibleto study the microscopic anatomy of translucent andsemi-opaque structures, such as the conjunctiva.9

A systematic examination of the microscopicanatomy of the conjunctiva in vivo will allow confir-mation of the results and observations of earlier invitro light and electron microscope studies and mayalso reveal new insights into this tissue structure. Aswell, such a study will generate valuable baselineinformation against which pathological, surgical,drug- or contact lens-induced changes can beappreciated. The aim of this study, therefore, is toundertake a qualitative and quantitative investiga-tion of the cellular anatomy of the normal humanbulbar conjunctiva using LSCM.

METHODS

Subjects

Eleven subjects (nine men and two women), aged27 � 6 years, agreed to participate in this studyafter the procedures were fully explained andconsent forms were signed. None of the subjectswore contact lenses or had a history of significantocular disease. Ethics approval for this study wasobtained from Queensland University of TechnologyHuman Research Ethics Committee (ApprovalNo. 0700000362).

Confocal microscopy

Subjects underwent examination with a LSCM; spe-cifically, we used a Heidelberg Retinal Tomograph(HRT3) equipped with a Rostock corneal module(RCM) (Heidelberg Engineering GmbH, Heidelberg,Germany). One eye of each subject (the eye preferredby the subject) was examined. The eye was ana-esthetized with 0.4% benoxinate hydrochloride(Chauvin Pharmaceuticals, Surrey, UK). It has beenshown previously that the anaesthetic used in thisstudy does not affect tissue structure as viewed withthe confocal microscope.10

Images of the bulbar conjunctiva were captured atfour cardinal locations relative to the cornea (superior,

inferior, nasal and temporal conjunctiva). To achievethis, the subject was instructed to direct their gazeextremely, in turn, towards the opposite direction tothe region of measurement. A disposable perspex capwith a flat anterior surface (the ‘TomoCap’) is fixed tothe front of the main objective element (the RCM). Adrop if GenTeal Gel (Carbomer 980, 0.2%, Novartis,North Ryde, NSW, Australia) is inserted into theTomoCap to form an optical coupling between thefront element of the RCM and the TomoCap. Anotherdrop of GenTeal Gel is placed on the front of theTomoCap to aid subject comfort.

The surface of the TomoCap was positioned on theconjunctiva such that the centre of the applanatingsurface was about 2–4 mm from the limbus. Thelocation that gave the best image of a continuousfield of cells close to the limbus was used. The orderof measurement at the four cardinal locations wasrandomized. For each location on the conjunctiva, atleast five good-quality digital images were capturedfrom each of the anterior and posterior conjunctivalepithelium and the underlying conjunctival stroma.Images were also captured of any other interesting,unusual or unidentified features. All images werescrutinized and representative images of goodquality were selected for qualitative evaluation andpresentation. The aforementioned procedures wereconducted on all 11 subjects.

Morphometry

The thickness of the epithelium was determined byfocusing the instrument on the anterior conjunctivalsurface, noting the depth measurement readout(‘baseline value’), and focusing through to the junc-ture of the basal conjunctival epithelium and theunderlying connective tissue. At this point, a notewas taken of the depth measurement readout; thisvalue was subtracted from the baseline value to givethe thickness of the epithelium. This procedure wasrepeated three times at each cardinal location on eachsubject, and the average of the three determinationswas calculated for each subject and cardinal site.

Cell density was determined using the HRT3/RCM cell count software. The three clearest imagesof each of the superficial and basal epithelial layerswere identified and stored for analysis. In eachimage, a clear continuous area of cells was selectedand at least 50 cells were counted. The mean of threedeterminations was calculated for each subject todetermine the cell density of each layer and cardinallocation.

Goblet cells were counted by selecting, for eachsubject and cardinal location, the clearest image fromany layer of the epithelium with the greatest numberof visible cells by inspection. The number of cells inthe complete 400 ¥ 400 mm2 image area was counted,

336 Efron et al.


and GCD computed. The density of mature forms ofLangerhans cells (LCD) was determined in the samemanner as mentioned earlier, with the exception thatthe images with the greatest number of cells, byinspection, in the epithelium or stroma wereassessed.

Popper et al. have reported that confocal micros-copy can produce repeatable quantitative measure-ments of tissue cell density with intersession andintrasession repeatabilities of 8.3% and 5.8%, respec-tively, using semiautomated counting techniquessuch as those used in this study.11 Three repeatedmeasurements at three sessions revealed an intrases-sion coefficient of variability for epithelial thicknessof 4.3%, and an intersession and intrasession coeffi-cient of variability for bulbar superficial epithelial celldensity of 4.7% and 3.2%, respectively.

Statistical methods

The significance of any differences in epithelialthickness and cell densities between the four cardi-nal locations was tested using an analysis ofvariance. If significant differences were detected, apost-hoc analysis was applied to determine whichspecific locations were different. The significance ofany differences between superficial and basal epithe-lial cell density at the four cardinal locations wastested using the Student’s t-test for matched pairs. AP-value of less than 0.05 was considered significant.

RESULTS

Qualitative analysis

Images were obtained from the right eye of eightsubjects and the left eye of three subjects. A typical

appearance of the superficial epithelium is shown inFigure 1a. Light to medium grey round nuclei arevisible in a uniform field of cells. The cytoplasm isgenerally dark grey, but a very faint light grey cellborder is sometimes visible. In many subjects, all orpart of the cell nuclei appear to be bright white(Fig. 1b). In a few images, large cells with a light-coloured cytoplasm and bright white nucleusare seen overlying the superficial epithelium(Fig. 1c).

Cells in the middle and posterior layers of theepithelium are observed as having a light greyborder (Fig. 2a). The pallor of the cytoplasm of mostcells is uniformly grey. Nuclei are not visible. Thecells vary in size and shape, and have three to sixstraight or curved sides. In some instances, greatervariation in cell size (polymegethism) and shape(pleomorphism) is observed throughout the field(Fig. 2b).

Significantly different morphology is observed insome subjects. In Fig. 2c, a mass of bright white dotswas observed throughout the posterior epithelium.These dots generally appear to lie within cellborders, and in some instances appear to befragmented. In Figure 2d, large and clearly delin-eated portions of the field contain cells that have abright, granular cytoplasm, with bright cells bordersvisible throughout most of these areas.

A myriad of morphological features are observedin the conjunctival stroma. The typical appearance ofthe adenoid layer lying immediately posterior to theepithelium is a granular field of varying pallor, withan assortment of fibrous and linear features andbright white and grey dots, the latter presumablyrepresenting cellular elements (Fig. 3a). The deeperfibrous layer of the conjunctival stroma contains

a b c

Figure 1. Superficial epithelium of bulbar conjunctiva imaged using laser scanning confocal microscope. (a) Common appearance ofsuperficial epithelium. (b) Less common appearance of superficial epithelium with brightly reflective nuclei. (c) Large cells with a lightlyshaded cytoplasm and bright white nucleus (arrows) are seen overlying the superficial epithelium. These may be corneal epithelial cellsthat have sloughed off the cornea and washed over the conjunctiva. (All images 400 ¥ 400 mm).

Confocal microscopy of the bulbar conjunctiva 337


extensive meshwork patterns of fibrous tissue, withbundles of fibrous strands running straight orslightly curved paths in various directions (Fig. 3b).

Occasional blood vessels of varying diameter canbe seen throughout the fibrous layer of the stroma.These appear as a broad black linear or slightlycurved structure with parallel sides. Small reflectivecells with a light grey cytoplasm, and sometimeswith a bright nucleus, are seen within the vessellumen (Fig. 3b). Figure 3c shows a large vessel andsmaller branch, both of which are filled with cellularelements. A branching network of vessels lyingwithin fibrous connective tissue is shown inFigure 3d.

Round black circles of various sizes were observedin the superficial epithelium of six subjects, typicallysurrounded by a rosette arrangement of cells(Fig. 4a). Some cells in the deeper layers of the epi-thelium appear slightly larger and paler than sur-rounding cells, and in many cases the light greycytoplasm of these cells can not be distinguishedfrom the cell border (Fig. 4b). These cells sometimesform in clusters (Fig. 4c).

At various depths within the epithelium, largeblack inclusions, referred to as ‘conjunctival micro-

cysts’, were observed in two subjects (Fig. 5a).These are typically round or oval in shape and havea bright white or grey border. Small, white or greyspherical elements and other faint material can beseen inside some of these microcysts. From a three-dimensional perspective, the microcysts appear tobe spherical or cigar-shaped; this can be deducedfrom the serial sections through a microcyst asshown in Figure 5b.

Clusters of white cells are observed at variouslayers of the epithelium and in the stroma. Thesecells, which are thought be mature forms of Langer-hans cells, are comprised of short, feathery, whitelinear branches or extensions, often forming aY-shaped pattern. One or two small, bright dots,slightly wider than the branch arms, typically appearsomewhere along the cell. This is often referred to asa ‘dendritic’ appearance. Figure 6a shows a group ofLangerhans cells within the epithelium. The cellsappear to encircle some epithelial cells in Figure 6b.In Figure 6c, Langerhans cells are seen at the level ofthe conjunctival stroma.

Whereas the cell bodies of two Langerhans cellsare clearly visible in Figure 6b, the cell borders ofmany of the cells in this figure can be seen to

a b

c d

Figure 2. Basal epithelial layersof bulbar conjunctiva imagedusing laser scanning confocalmicroscope. (a) Common appear-ance of basal epithelial layer. (b)Less common appearance of basalepithelial layer with cells displayingsignificant polymegethism andploemorphism. (c) Bright whitereflective elements lying within theborders of cells in the basalepithelium. (d) Unusual image ofbasal epithelium, with largeregions of the field containing cellswith a bright, granular cytoplasm.(All images 400 ¥ 400 mm).

338 Efron et al.


contain, or be partly composed of, fine whitebroken or continuous linear segments. Thesemay represent fine nerve endings surroundingthe basal epithelial cells, although further his-tochemical studies would be required to confirmthis.

Inspection of images taken from the four cardinalsites of the bulbar conjunctiva failed to reveal anyobvious or consistent differences in morphology ofany of the tissue layers or cellular elements (asidefrom differences in cell counts; see ‘Quantitativeanalysis’ below).

a b

c d

Figure 3. Bulbar conjunctivalstroma imaged using laser scan-ning confocal microscope. (a)Adenoid layer, immediately poste-rior to the epithelium, with somefibrous elements, linear featuresand bright white and grey dots. (b)Deeper fibrous layer of the stroma,comprising of a connective tissuenetwork of dense fibre bundles. Acurved blood vessel filled with cellsis visible to the right of the field. (c)Large vessel and branch filled withcellular elements, in the fibrouslayer. (d) Branched vessel lyingwithin a bed of fibrous connectivetissue in the fibrous layer. (Allimages 400 ¥ 400 mm).

a b c

Figure 4. Goblet cells in the conjunctival epithelium imaged using laser scanning confocal microscope. (a) Orifices of goblet cells(arrows) in the superficial epithelium that have expelled their contents. (b) Goblet cells (arrows) are slightly larger and paler thansurrounding non-secretory epithelial cells. (c) Cluster of goblet cells in the upper left of the field may represent a portion of a goblet cellcrypt. (All images 400 ¥ 400 mm).



Quantitative analysis

The mean thickness of the bulbar conjunctival epi-thelium is 32.9 � 1.1 mm, with the superior bulbarconjunctiva being slightly thinner than at the otherthree cardinal locations (Table 1). The overall densityof superficial bulbar conjunctival cells (2212 � 782cells/mm2) is about 7% lower than that of basalbulbar conjunctival cells (2368 � 741 cells/mm2)(P < 0.0001) (Table 2). The density of superficialbulbar conjunctival cells is also significantly lowerthan that of basal bulbar conjunctival cells at tempo-ral, nasal and superior locations.

There is no significant regional variation in super-ficial bulbar conjunctival cell density; however, basalbulbar conjunctival cell density was dependent onlocation (P � 0.0001). Post-hoc analysis revealed theinferior region to have a significantly lower celldensity than the other three cardinal locations.

Overall GCD and LCD were determined to be111 � 58 and 23 � 25 cells/mm2, respectively(Table 3). There is a significant variation in GCDbetween cardinal locations (P < 0.001), and post-hocanalysis revealed nasal GCD to be significantlygreater than at other locations. The superior regionshowed the lowest density of goblet cells (only threeof 11 subjects had goblet cells visible) and maturedendritic Langerhans cells were not observed in thisregion in any subjects.

DISCUSSION

Qualitative observations

Bron et al. describe the anatomy of the bulbar con-junctiva as being composed of two main structures: asuperficial epithelial layer of about five to sevenlayers thick, and an underlying conjunctival stroma.1

Lawrenson maintains that the stroma can be resolvedby light microscopy into two distinct layers: a super-ficial adenoid layer containing lymphocytes andmast cells, and a deeper and thicker fibrous layercontaining the majority of conjunctival blood vesselsand nerves (Fig. 7).12 Posterior to the conjunctiva isthe denser connective tissue of Tenon’s capsule(fascia bulbi), and posterior to this is the episcleraand then sclera.

Observations of the human bulbar conjunctivausing the LSCM in this study are consistent with theknown histology of this tissue as revealed by in vitrostudies using light and electron microscopy.1,12,13 Ourobservations are also broadly consistent with theLSCM observations of the bulbar conjunctiva byother authors,14–17 with one exception. Messmer et al.claim that figure 1A of their paper shows the normalsuperficial bulbar epithelium;14 however, this imageshows large polyhedral cells of varying reflectivity,which are characteristic of the corneal superficial

a

b

Figure 5. Conjunctival microcysts imaged using laser scanningconfocal microscope. (a) Microcyst within the conjunctival epi-thelium (arrow), with pale grey border and containing whatappears to be discrete round white mass of cellular material.Some more dispersed faint grey material can be seen inferior andto the left of the white mass. (400 ¥ 400 mm). (b) Serial sectionthrough microcyst containing small cellular elements; imagesbeginning top left progress from anterior to posterior basal epi-thelial layers in approximately 4 mm steps. This suggestsan approximately spherical or cigar shape. (All images108 ¥ 108 mm).

340 Efron et al.


epithelium.9 The images of the superficial bulbarepithelium displayed in the present paper, and byKobayashi et al.,15,16 are distinctly different. The cellsare much smaller and more densely packed. A likelyexplanation for the observation of Messmer et al.14 isthat, in the course of imaging the conjunctiva, theobjective of the confocal microscope inadvertentlyslipped momentarily onto the cornea, which we havefound can occur in patients with unsteady fixation.The large superficial cells with a light-coloured cyto-plasm and bright white nucleus displayed infigure 1C of Messmer et al.14 – against a backgroundof what is unmistakably the superficial bulbar con-junctiva – may represent superficial corneal epithe-lial cells that have sloughed off the cornea, washedonto the conjunctival surface and been captured bychance in this image.

The small bright dots that can be observed scat-tered throughout some images, such as Figure 3a, arelikely to represent one or more of the variety of celltypes that are known to reside in the conjunctiva.13

However, the LSCM lacks the resolution to differen-tiate most of these cells on the basis of morphology,and cell identification via histological staining is notpossible when observing human tissue in vivo.

Certain cells can nevertheless be identified on thebasis of their characteristic form. For example, thewhite dendritic cells observed at various layers ofthe epithelium and in the stroma are consistent withknown descriptions of mature Langerhans cellsbased on observations using light microscopy.1 Cellsof almost identical appearance to those illustrated inFigure 6 have also been published by Messmeret al.14 and Kobayashi et al.15 Immature, non-dendriticLangerhans cells are equipped to capture antigens,while mature dendritic forms are able to sensitizenative T cells through major histocompatibilitycomplex molecules and secretion of interleukin-12as well as costimulatory molecules, and thus repre-sent an integral part of the immune system.18

Interspersed throughout the bulbar conjunctivalepithelium are goblet cells, which are responsible forproducing mucin – an important component of thetear film that is partially responsible for keeping theocular surface moist.1,12,13,19 Goblet cells are notvisible in the superficial epithelium when usingLSCM, although we have found indirect evidence oftheir presence in this layer. Figure 4a shows nineblack circles of various sizes on the surface of theconjunctival epithelium. These circles may representholes or openings in the surface tissue, and could bethe openings of goblet cells that have expelled, or arein the process of expelling, their mucin content.

In Figure 4b, which is an image of a deeper epi-thelial layer, the cytoplasm of a number of cells issomewhat lighter and larger than that of cells in theremainder of the field. Our images of these cells are

a b c

Figure 6. Mature forms of Langerhans cells displaying characteristic dendritic processes, seen in the bulbar conjunctiva imaged usinglaser scanning confocal microscope. (a) Langerhans cells within the superficial epithelium. (b) Langerhans cells in the basal epithelium(long arrows). Small continuous and broken sections around cell borders (short arrows) may represent nerve endings. (c) Langerhans cellswithin the conjunctival stroma. (All images 400 ¥ 400 mm).

Table 1. Bulbar conjunctival epithelial thickness†

Conjunctival location Thickness(mm)

Temporal 33.7 � 1.4Nasal 33.2 � 1.0Superior 31.7 � 1.3‡

Inferior 33.0 � 1.7Overall 32.9 � 1.1Significance of difference§ F3,10 = 10.36

P < 0.0001

†Mean � standard deviation. ‡Post-hoc analysis reveals thatthis value is significantly lower than the values for other locations.§Excluding ‘Overall’, which is a mean of the four locations.



consistent with those of previous investigators whohave used LSCM to assess goblet cells in the bulbarconjunctiva. Rath et al. have demonstrated that gobletcells can be easily identified in the deeper layers ofthe human bulbar conjunctiva using LSCM, beinglarger and of a lighter shade than the surroundingnon-secretory epithelial cells.17 Kobayashi et al. havepublished a similar image in which slightly largercells with lighter cytoplasm are identified as gobletcells.15 Messmer et al. show a deeper layer of bulbarconjunctival epithelium in which two oval cells withdark cytoplasm, which are two to three times the sizeof surrounding cells, are identified as being gobletcells.14 Rath et al. suggest that variation in the shadeof the cell cytoplasm as viewed using confocalmicroscopy may correlate with various secretion con-tents or suggest distinct, recognizable, functionalconditions (hypo- or hypersecretion).17

The appearance of goblet cells using LSCM is con-sistent with histological evidence of whole mountsand cross-sections of excised human bulbar conjunc-tiva that have been stained to reveal mucin-

containing cells.19 As can be seen in the lightmicrograph of human bulbar conjunctival epithe-lium (Fig. 7), fewer goblet cells reside in the deeperlayers of the epithelium compared with the epithe-lial surface. Indeed, Kessing made this very observa-tion in his seminal work.19 The close gathering ofcells of lighter shade in Figure 4c may represent agoblet cell crypt, which is described as a dense accu-mulation of goblet cells extending from the epithe-lium into the conjunctival stroma.1,12,13,19

We are apparently the first to have observedmicrocysts in the bulbar conjunctival epithelium ofnormal subjects. Messmer et al. have reported theappearance of microcysts in the palpebral conjunc-tiva; the microcysts shown in figure 2A of their paper

Table 2. Bulbar conjunctival epithelial cell density†

Conjunctival location Superficial cell density(cells/mm2)

Basal cell density(cells/mm2)

Significance ofdifference

Temporal 2187 � 763 2429 � 847 t = -2.74P = 0.0210

Nasal 2306 � 792 2457 � 791 t = -2.74P = 0.0210

Superior 2110 � 881 2437 � 723 t = -4.75P < 0.0010

Inferior 2245 � 774 2148 � 686‡ t = 0.95P = 3.600

Overall 2212 � 782 2368 � 741 t = -5.28P < 0.0001

Significance of difference§ F3,9 = 0.52 F3,9 = 11.41P = 0.6700 P < 0.0001

†Mean � standard deviation. ‡Post-hoc analysis reveals that this value is significantly lower than the values for other locations of thebasal conjunctiva. §Excluding ‘Overall’, which is a mean of the four locations.

Table 3. Bulbar conjunctival goblet and Langerhans celldensity†

Conjunctivallocation

Goblet cell density(cells/mm2)

Langerhans cell density(cells/mm2)

Temporal 49 � 69 43 � 42Nasal 262 � 166‡ 32 � 38Superior 7 � 12 0 � 0Inferior 124 � 111 17 � 49Overall 111 � 58 23 � 25Significance of

difference§F3, 40 = 12.34 F3, 39 = 2.41

P < 0.001 P = 0.082

†Mean � standard deviation. ‡Post-hoc analysis reveals thatthis value is significantly higher than the values for other loca-tions for goblet cell density. §Excluding ‘Overall’, which is a meanof the four locations. Figure 7. Light micrograph of the bulbar conjunctiva. The epi-

thelium (e) is four to eight cells thick. The stroma can be differ-entiated into adenoid (a) and fibrous (f) layers. Goblet cells stainhere as a lighter shade and can be seen primarily in the superficialepithelium (long arrows), with fewer visible in the basal epithe-lium (short arrows) (stain: 1% toluidine blue; image width:720 mm). Courtesy of Professor John Lawrenson.

342 Efron et al.


are identical in appearance to those illustrated inFigure 5a of this paper.14 Messmer et al. suggest thatconjunctival microcysts may represent occludedgoblet cells with retention of their contents.14

Indeed, Kessing reported that such inclusions were acommon finding with increasing age,19 and the lightmicrograph of this phenomenon that he published issimilar in appearance to the microcysts observed inthis study and by Messmer et al.14 Interestingly, Cian-caglini et al. believe that conjunctival microcysts arepathognomonic of ocular hypertension and glau-coma, and are not observed in normal healthy sub-jects;20 the observation of microcysts in healthysubjects reported here and by Messmer et al.14 chal-lenge the observation of Ciancaglini et al.20

According to Bron et al., conjunctival nerve fibreslose their myelin sheaths and form a subepithelialplexus in the superficial substantia propria.1 Theythen form an intra-epithelial plexus around the basesof the epithelial cells, sending free fibrils betweenthem. Certainly, the human cornea contains an exten-sive nerve plexus immediately posterior to the epi-thelium – the sub-basal nerve plexus – that is clearlyvisible using confocal microscopy.9,21 However, sucha nerve plexus was not observed in the bulbar con-junctiva in this study. A possible explanation is thatthe individual nerve fibres may be much smallerthan in the corneal sub-basal nerve plexus, and aretherefore unable to be resolved. The fine white linearelements surrounding basal conjunctival epithelialcells in Figure 6b may represent anterior projectionsfrom a nerve plexus that forms nerve endings thatsurround these cells. No other evidence of nervefibres could be found in our database of conjunctivalimages, and neither Messmer et al.14 nor Kobayashiet al.15 reported observing nerve fibres in the bulbarconjunctiva.

Quantitative observations

The overall thickness of the bulbar conjunctival epi-thelium reported here (32.9 � 1.1 mm) is almostidentical to that reported by Messmer et al.14 (32 � 9.6 mm). Thus, the conjunctival epithelium is about40% thinner than the corneal epithelium(52 � 3 mm).22 We have found the superior bulbarconjunctival epithelium to be thinner than that at theother three cardinal sites assessed. This may be dueto pressure against the globe (and hence the conjunc-tiva) by the upper lid, which is perhaps greater thanthat of the lower lid.23

Light and electron microscopy reveals that thetransition in tissue morphology from conjunctivalstroma to fascia bulbi, episclera and sclera is more orless continuous1,12,13 and no clear demarcations canbe discerned; this was clearly also the case with theconfocal microscope. It is therefore not possible to

determine the overall thickness of the conjunctivausing LSCM with any degree of certainty.

The cell densities of the superficial and basalbulbar conjunctival epithelium reported here (2212� 782 and 2386 � 741 cells/mm2, respectively) areabout half of the values calculated from the cell areadata of Messmer et al.14 (4708 and 4545 cells/mm2,respectively). Given that the same model of confocalmicroscope was used in both studies, the reason forthis discrepancy is unclear. Whereas Messmer et al.reported that there was no statistically significantdifference between superficial and basal cell den-sity,14 the present study established that basal celldensity is significantly greater than superficial celldensity overall, and is greater at temporal, nasal andsuperior locations. By way of comparison, superficialand basal epithelial cell density of the cornea deter-mined using confocal microscopy is 1213 � 370 and5699 � 604 cells/mm2, respectively.24

The values of GCD reported here (overall mean of111 � 58 cells/mm2) are somewhat lower than thosereported by others. For example, from the lightmicroscopy data of Kessing19 it can be determinedthat overall GCD in the bulbar conjunctiva of sub-jects aged 27 years (the mean age of the subjects inthe present study) is about 570 cells/mm2.Rodriguez-Prats et al., using impression cytology,found GCD of normal bulbar conjunctiva to be424 � 105 cells/mm2.7

The discrepancies highlighted earlier may beattributed to the stark differences in the methodolo-gies used to estimate GCD. Kessing examined wholemounts of cadaver or live human biopsy samples, andhe acknowledged that this process can lead to arte-facts and distortions of tissue dimensions.19 Thus, forexample, tissue shrinkage would result in a highercell counts per unit area than the true value. Also,Kessing focused through all layers of the epitheliumand counted goblet cells observed at every level.19

This approach contrasts with the present LSCM studywhereby cells were only counted in a single plane.Cell counting using LSCM would therefore lead to alower result than that which would be obtained byexamining the same tissue sample using light micro-scopic through focusing.19 Notwithstanding the afore-mentioned factors, the observation of Kessing19 thatGCD is the lowest in the superior temporal quadrantand the highest in the inferior nasal quadrant isentirely consistent with the data reported here. Vujk-ovic et al. also reported an unequal distribution ofGCD in the bulbar conjunctiva.25

Impression cytology involves obtaining a ‘surfacebiopsy’ from the conjunctiva.4 As with whole mounttechniques,19 the extracted tissue sample may changedimensions during biopsy, staining and tissuemounting, possibly introducing artefact that canaffect in cell counts.



Steuhl et al. undertook a quantitative analysis ofLCD on excised human conjunctival biopsy speci-mens using the Langerhans-specific anti-CD1a anti-body and found LCD in the superior temporal zoneof the bulbar conjunctiva to be 1.0 cells/mm2.26 UsingLSCM, we were unable to detect Langerhans cells inthe superior bulbar conjunctiva, but recorded a LCDof 43 � 12 cells/mm2 in the temporal bulbarconjunctiva. Again, differences in methodologiesmay account for the discrepant cell counts. Interest-ingly, our estimates of LCD in the bulbar conjunctivaare broadly consistent with that in the central humancornea, which was determined using LSCM byZhivov et al.18 to be 34 � 3 cells/mm2.

A limitation of the present study is the impreci-sion of the sampling location, which may have ledto some variability of the data, particularly thatbetween conjunctival regions. In future, precise ana-tomical localization of the conjunctival regions willfacilitate a more precise and repeatable characteriza-tion of various regions of the conjunctiva.

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in vivo confocal microscopy of the bulbar conjunctiva

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