To date there are around 900 published papers dealing with in vivo CM of the cornea (source: Pubmed, April 2008). The following summarized results are in line with the known histology of the cornea and have been reported in recent publications.16–20
Superficial cells (Fig. 2a) are characterized by a polygonal cell pattern, bright illuminated cytoplasm, reflecting nucleus and perinuclear dark halo. Cell size is up to 50 µm in diameter and about 5 µm thick with individual variations.21 The average density in the corneal centre and periphery is from about 624 cells/mm2.22 to 1213 cells/mm2.23
Figure 2. In vivo confocal images of the normal cornea. (a) Superficial cells. (b) Upper wing cells. (c) Low wing cells. (d) Basal cells. (e) Sub-basal nerve plexus. (f) Bowmane's membrane. (g) Anterior stroma. (h) Posterior stroma. (i) Endothelium. (j) Oblique section through corneal epithelium and anterior stroma – all layers are present. (k) Three-dimensional (3D) reconstruction of the whole cornea (see 3D reconstruction section).
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The cells of the epithelial intermediate layer, or wing cells (Fig. 2b,c), form a regular mosaic with sharp and reflecting cellular borders. The wing cells are smaller in size (about 20 µm) but regular in form. They can also be subdivided into upper (Fig. 2b) and lower (Fig. 2c) wing cells; the latter are smaller.19 To date there are only a few studies reporting the cell count in this layer.24 The average density is 5000 cells/mm2 in the corneal centre and 5500 cells/mm2 in the periphery.24
Basal epithelial cells (Fig. 2d) have a smaller diameter (8–10 µm) and appear as a layer of cylindrical cells where nuclei cannot be remarked by a reflecting border. The average density varies from about 600023 to 900025 cells/mm2 in the corneal centre and 10 000 cells/mm2 in the periphery.24
The ratio between superficial, intermediate and basal cells (accordingly to the cell density) is approximately 1 : 5 : 10,24 whereas another two studies report ratios between the superficial cells and basal cells of 1 : 523 and 1 : 7.25 The difference in cell ratios in these studies was interpreted by the strategy to study the lowest layer of basal cells situated directly over the Bowman's membrane, where the highest cell density is expected.24 There are no published data about cell count changes caused by age in any cell layer of the epithelium.
The sub-basal nerve plexus (SNP) (Fig. 2e) is characterized by the presence of hyper-reflective fibres of 4–8 µm length,26–29 connected with anastomoses and organized in a vortex pattern in the lower nasal quadrant of the paracentral cornea. A two-dimensional reconstruction of the SNP maybe performed, allowing mapping of the central 5 mm of the cornea.30
The Bowman's layer (Fig. 4f) is an 8–10 µm thick zone consisting of randomly arranged collagen fibrils located in between the basal cells and the stroma. Moreover, in vivo CM shows polymorphic structures composed of fibrillar materials (K-structures) beneath the Bowman's layer in normal human subjects. It was presumed that these microstructures (5–15 µm in diameter) might correspond to the modified and condensed anterior stromal collagen fibers/lamellae that merge into the Bowman's layer and that these fibrillar materials may be responsible for the formation of the anterior corneal mosaic.31,32
Figure 4. Phases of development of the corneal ulcer. (a, b) Progressive phase: oedema of epithelium and stroma: (a) swelling of superficial cells, note the border of the ulcer region (arrow); (b) oedema of wing and basal cells (*), infiltration with inflammatory cells (leucocytes and Langerhans cells – arrow). (c,d) Regressive phase: (c) reconstruction of oblique section – minimal epithelial oedema, all epithelial layers are visualized, inflammatory cells at the level of the SNP (arrow), development of scar tissue (*), intact stroma (#); (d) ulcer edge (arrow) wit inflammatory cells. (e) Healing phase, ulcer edge: intact stroma with a normal keratocyte pattern (*), scar tissue formation at the ulcer edge (arrow) and regular pattern of the regenerated wing cells (#) are presented. Depth difference between the stroma (*) and wing cells (#) is about 40 µm.
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Corneal stroma (Fig. 2g,h) forms around 80–90% of the whole corneal volume. It consists of three main histological components: cellular, acellular and neurosensorial. The keratocyte nuclei are visible as egg-shaped reflecting light corpuscles, whereas the connective lamellae appear black (that is transparent) because of their optic properties. Patel et al. presented a review of 18 studies of keratocyte density in normal corneas.20 Taken together, keratocyte density is highest in the anterior stroma, clearly declines toward the central stroma, and increases again slightly in the posterior. The density of keratocytes decreases with age.33,34 The stromal nervous fibres, which are thicker than the subepithelial ones, run along the stromal tissue along a straight pathway, although it is sometimes possible to find dichotomous branches (T and Y shapes).19
Descemets membrane is a thin (6–10 µm) homogeneous layer, located in between the posterior stroma and endothelium, and is not visible with CLSM.
The endothelium (Fig. 2i) is a monolayer of cells arranged in a hexagonal pattern of honeycomb regular mosaic, where the cells are normally identical in size and shape, that is, without signs of polymegatism and pleomorphism. Sometimes it is possible to visualize the nucleus of the cells. The total number of cells is about 500 000 in a healthy subject with a normal cell density of 2500–3000 cell/mm2.19,24 The cell density decreases with age.35
Confocal laser scanning microscope enables not only en face imaging but also oblique cuts through several layers, offering a direct parallel to the traditional histological images (Fig. 2j).
Light scattering phenomenon determines the reflectivity of cells. The main factors influencing on the interaction of light beam and its transmission and absorption are, according to Bochard, cellular organelles and membranes, microvilli, microplicae and glycocylix.36,37 It was postulated that the presence of microdesmosomes in the epithelial layers could explain why the cell membranes of epithelial cells were more brightly illuminated (Fig. 2a,d) than those of the endothelial cells (Fig. 2i).
Confocal laser scanning microscope enables the differential diagnosis of different pathogens and, so far, the differentiation between bacterial, fungal or protozoan agents (Fig. 5).
Figure 5. Confocal microscopy and differential diagnosis of the ulcer. (a) The ulcer bottom is presented with hyper-reflective surface without any additional information. (b) Bacterial ulcer: massive infiltration with inflammatory cells (leucocytes and dendritic cells). (c) Protozoan ulcer: typical hyper-reflective Acanthamoeba cysts with up to 20 µm diameter located in the epithelium (arrow) (c1) as well double-walled structure (c2) located in the stroma. (d) Fusarium solani keratitis: hyper-reflective linear hyphae in the stroma (arrow) (courtesy of A. Labbe/Paris).
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Bacterial ulcers show as typical hyper-reflective defects without the structure of the ulcer bottom being recognizable (Fig. 5a). The adjacent epithelium is oedematous. The basal cell level as well as the SNP is infiltrated with leucocytes and LCs (Fig. 5b). The pathogen art differentiation is not possible.
Viral keratitis (herpes keratitis, epidemic keratoconjunctivitis) can be easily and accurately diagnosed by the presence of wire netting of the SNP fibres and dendritic cells.16,43 In the acute phase of the pathological process, LCs are located at the level of the lower intermediate cells, basal cells and nerve plexus. The decrease in dendritic cell density is a clear, indirect sign of recovery, a sign that can be used in clinical practice.
Quick and reliable identification of Acanthamoeba is one of the most acknowledged applications of CM in clinical practice.16,19,20,44–46 The typical structure of Acanthamoeba cysts and trophozoites allows rapid diagnosis and effective treatment. The cyst forms are presented as highly reflective, round-shaped particles 10–20 µm in diameter within the corneal epithelium and stroma (Fig. 5C). It is possible to find cysts as single, round-shaped structures, (Fig. 5C-1) or double-walled structures (Fig. 5C-2) as well as conglomerates.
Mathers et al.46 compared the results of CLSM with the polymerase chain reaction, and in 77% the diagnosis was correct. So far the combination of clinical experience and expert knowledge allows immediate diagnostic and appropriate therapy.
Diagnosis of fungal keratitis is the other key advantage of CM (Fig. 5d). Nevertheless, corneal scrapings for smears and cultures are still the primary diagnostic tool. CLSM is able to reveal numerous hyper-reflective elements resembling Fusarium, Aspergillus hyphae or Candida pseudofilaments in the anterior stroma even in the early phase of the disease.19,38,47,48 These findings are often associated with the presence of inflammatory cells (leucocytes as well as LCs). Brasnu et al.48 compared in vivo CM findings with the results of corneal smears and culture evaluation, and proved that using CLSM the clinician is able to determine the fungal agents. Moreover, it offers the in vivo possibility of fungal differentiation. Fusarium solani typically displays the presence of multiple highly reflective linear formations (hyphae) up to 300 µm in length and 5 µm in width, with branches at 90° angles in the anterior stroma. In contrast to hyphae, the Candida pseudiphilaments are characterized by numerous hyper-reflective particles from 10 to 40 µm in length and from 5 to 10 µm in width located in the anterior stroma. When the fact that invasive methods, such as the culture of corneal scrapings and biopsy specimens or PCR, have different grades of sensitivity and the fact that only about 25% of results are positive to the second week are taken into consideration, CLSM can be considered a rapid and reliable clinical diagnostic tool.
One of the main questions is the sensitivity and specificity of the CM. Kanavi et al.49 compared the results of corneal and/or contact lens case smear and culture in the diagnosis of infectious keratitis with the CM findings. Corneal and/or contact lens case smear and culture were positive in over 50% of eyes, including 40 cases of bacterial, 16 cases of fungal and 15 cases of acanthamoeba infection. CM revealed the pathogens in about 40% (50 eyes); 27 eyes with hyphae-like structures and 23 cyst and/or trophozoite-like structures. The sensitivity and specificity of confocal scans were 100% and 84% for the diagnosis of acanthamoeba keratitis versus 94% and 78% for fungal keratitis, respectively.
So far, in vivo CLSM is a rapid and reliable clinical tool for the diagnosis of acanthamoeba and fungal keratitis with high sensitivity and specificity compared with traditional smear and culture. Nevertheless, clinical situation and the experience of the ophthalmologist also play a decisive role in the treatment of the patient.
Contact lens wear
Distinct changes in corneal morphology, pachymetry and structure in contact lens wearers can be demonstrated by in vivo CLSM.
Epithelial changes, such as compression of the superficial cells, formation of mucin balls over the complete epithelial thickness by hydrogel contact lenses with subsequent activation of keratocytes, have already been discussed elsewhere.56 Increase of LC density as a reaction of the corneal surface (and the whole eye associated lymphatic tissue system as well) allows speculation about the changes in the immune status of the cornea. Both mature and immature forms of LC are typically present at the level of wing and basal cells.41 Moreover, an increased number of rolling leucocytes in the limbal vessels has been described by Efron.56 Continuous mechanical stimulation of the ocular surface, applanation of different contact lens storage solutions, different grades of oxygen transmissibility all change the immune status of the cornea and increase the risk of infection.
It has been reported that over 50% of contact lens wearers have LCs in their central and peripheral corneas.41 When compared with healthy volunteers, the LC densities of contact lens wearers are significantly higher in both the central (78 ± 25 cells/mm2) and the peripheral cornea (210 ± 24 cells/mm2), whereas the gradient of LC density from peripheral to central cornea was found to be almost identical in both groups. In the central cornea, LC density significantly decreased with the duration of contact lens wear.16,41
Typical changes observed in the stroma are the presence of hyper-reflective panstromal microdot deposits.16 An increased number of microdot opacities compared with the non-lens wearing eye is apparent,6,57 and has been associated with the duration of contact lens wear. These microdots are thought to be granules of lipofuscin-like material.
All cell layers (superficial, intermediate and basal cells) are present and characterized by bright cell borders and uniformly dark cytoplasm. The cell count increases with layer depth because of a decrease in cell diameter. Ladage has postulated that contact lens wear stimulates the proliferation of basal cells, slows desquamation of superficial cells and inhibits the turnover of the corneal epithelium.58,59
Eckard et al. quantified the changes in the epithelium, which can be summarized as follows: cell bodies of superficial cells are generally smaller (30 µm in contact lens wearers and up to 50 µm in the normal cornea).19 A significant increase in superficial cell density existed both centrally and peripherally. Structures of intermediate and basal cells were found to be identical to the normal probands. The cell counts of both cell types were significantly reduced only in the periphery. Corneal thickness in the corneal periphery decreased in proportion to the duration of contact lens wear. Age-related changes in cell count or epithelial thickness were not found. Stromal thickness was reduced in elderly contact lens wearers.
Signs of polymegethism, pleomorphism and endothelial precipitates are the most common findings in the corneas of contact lens wearers.16,19,57
Collagen cross-linking with riboflavin and UVA light is considered to prevent or delay the progression of keratoconus.60,61 CLSM enables visualization of the photopolymerization effect,62,63 as well as possible complications of the procedure.
Rarefaction of keratocytes in the anterior and intermediate stroma, associated with the stromal oedema, was observed immediately after treatment.62 Our data show the appearance of the honeycombed anterior corneal stroma, but without the typical hyper-reflective keratocyte nuclei, just after cross-linking (Fig. 7). These structures are thought to represent activated keratocytes characterizing with the highly reflective cell nuclei with their cytoplasm and cytoplasmic processes. Complete repopulation of the keratocytes was found 6 months after the cross-linking. No endothelial damage (change of the endothelial cell count or morphology) was observed at any time.64
Figure 7. In vivo confocal laser scanning microscope of the cornea after cross-linking treatment (UVA + riboflavin) of progressive keratoconus (volume rendering and en face sections in different depths).
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Corneal refractive surgery
Refractive surgery is one of the most dynamic developing facet of modern ophthalmic surgery. It is of great importance to obtain the most reliable information about healing processes. Cellular changes are often not visible using the normal slit-lamp technique, which is why the CLSM offers a useful opportunity.19,20,65 Wound healing after photorefractive keratectomy (PRK) as well as laser in situ keratomileusis (LASIK) could be reliably controlled, and haze development, interface zone, activated keratocytes, reinnervation and possible complications, such as epithelial ingrowths or fibrosis, could be easily identified. Another useful measureable parameter is the real flap depth (Fig. 8).
Figure 8. Representative confocal microscopic images after laser in situ keratomileusis 12 month postoperatively. The central flap in a depth of 160–170 µm with high reflective particles in a depth field of 20 µm.
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It has been shown that real flap depth is thinner than the planned one, and that there are no changes in flap thickness over time.66,67 However, some studies have shown an increase in flap thickness.68 The depth of the flap can be easily quantified as the distance between the superficial cell layer and the interface zone. The latter is characterized by the presence of multiple hyper-reflective spots in the interface zone, as well as hyper-reflective (activated69) keratocytes.19 These signs of the interface zone decrease over time, but still present many years after LASIK66,70 and act as a landmark for CM. Until now, it has not been possible to determine the nature of this debris, which could consist of both organic and inorganic material (the residual materials of microkeratomy, secretion of meibomian gland, cellular debris).65 It has been speculated that haze originates from these activated keratocytes rather than from the extracellular matrix deposition. Moreover, a significant decrease in keratocyte density (the so-called acellular zones both sides of the interface) has been reported. Kaufman et al.65 described a decline in the keratocyte cell count within the flap zone during the 3-year follow-up period. These data could help to evaluate long-term corneal stability, refractive stability and cellular integrity after LASIK.
Reinnervation of the cornea is a cornerstone question in refractive surgery. CLSM allows easy detection of the SNP, and it is possible to track separate nerve fibres from the limbal area over the cut zone to the central cornea. The literature review shows contradictory data reporting that re-innervation in the central cornea starts somewhere between the first and sixth month. The SNP has completely recovered 2 years after PRK71 as well as after LASIK (our data), but even months after total restoration of corneal sensation the typical normal architecture of the corneal nerve anatomy could not be found. Our studies also show the possibility of complete regeneration of the whirlpool pattern after LASIK.
The other possible application of CLSM is the evaluation of haze. Haze can be described as the degree of backscattering of light by the cornea. Physiologically, it is explained by a keratocyte repopulation response with a higher density and reflectivity of migratory fibroblasts and myofibroblast transformation after repopulation.72,73
Imaging of the filtering bleb
One advantage of the recent advancement of CM is the ability to image the filtering bleb.74–77 It allows the analysis of bleb microstructures that are invisible under a slit lamp, as well as estimation of the bleb function. Thus, the epithelial microcysts, total stromal cyst area, absence of encapsulated stromal cysts and minimal vascularisation, as well as the absence of tortuous conjunctival vessels, are the signs of a good bleb function.76 The sub-epithelial connective tissue is widely spaced in functioning blebs, whereas the tissue is dense in non-functioning blebs.74,75 Moreover, Guthoff et al. classified the stromal structure into four patterns (trabecular, reticular, corrugated, compacted). The trabecular structure occurs only in functioning blebs, particularly in the early postoperative period. In contrast, corrugated, reticular or compact stromal patterns in early blebs tend to indicate a less favourable functional status.77