confocal microscopy examination of the normal corneal stroma. Keratocyte nuclei are visible as oval, round or egg-shaped hyper-reßective structures (Fig. 4.1d). Stromal nerves appear as hyper-reßective thick linear structures with sometimes visible dichotomous branches (Fig. 4.1e).

The Descemet membrane is observed as a thin (6Ð8 mm) amorphous and acellular layer located between the posterior stroma and the endothelium. This layer is not visible in young and normal subjects. The healthy corneal endothelium consists of a monolayer of hexagonal reßective cells arranged regularly in a honeycomb pattern with hypo-reßective cell borders (Fig. 4.1). Although IVCM images of the cornea consist of optical sections oriented parallel to the tissue, cross section may also be obtained. Not only the cornea, but also the conjunctiva and the limbus could be imaged with IVCM [1].

Clinical Applications

The early detection and diagnosis of infectious keratitis and the evaluation of corneal wound healing after refractive surgery or corneal surgery were the Þrst clinical applications of confocal microscopy. With improvement of in vivo confocal microscopes, these clinical applications were developed and new clinical applications have been demonstrated or are under development.

Infectious Keratitis

As an early diagnosis or even a high suspicion of an infective microorganism may lead to treating more rapidly and improve the outcome of infectious keratitis, it represents one of the most important clinical uses of IVCM. However, only the morphology of cells could be used to differentiate cell types and microorganisms with IVCM. As most bacteria have a size below or approaching the resolution limit of IVCM, these microorganisms could not be visualized directly using IVCM unless they presented distinctive features such as a larger size or a Þlamentous form [2]. Similarly to most bacteria, the virus size does not allow their direct visualization using IVCM. However, structural corneal changes could be observed and monitored in patients with bacterial or viral keratitis and differential diagnosis may beneÞt from this technique [2].

IVCM can visualize Þlamentous and yeast fungi within the cornea of patients with FK and may be helpful in managing this disease by providing a rapid diagnosis so that treatment can be initiated earlier [3, 4]. Fusarium and Aspergillus fungi appear as hyper-reßective interlocking Þlaments approximately 3Ð10 mm in width and 200Ð400 mm in length (Fig. 4.2aÐc). Some publications have also reported IVCM images of other fungi such as Beauveria bassiana, Candida Albicans, Microsporidia, Alternaria alternata, or Penicillium [2].

The clinical diagnosis of Acanthamoeba is often difÞcult because the early clinical presentation is nonspeciÞc. Contrary to viruses and bacteria, the size of Acanthamoeba cysts or trophozoites allows their direct visualization using IVCM. Numerous publications have described the use of IVCM as a noninvasive tool to

Fig. 4.3 In vivo confocal microscopy (IVCM) images (400 × 400 mm, Heidelberg Retina Tomograph Ð Rostock Cornea Module (HRT-RCM)) of corneal dystrophies. Microcysts in the basal epithelial layer in MeesmanÕs corneal dystrophy (a). Aberrant thickened basement membrane penetrating into the epithelium in epithelial basement membrane dystrophy (EBMD) (b). Granular hyper-reßective material within the basal epithelium and the BowmanÕs layer in Reis-Bucklers dystrophy (c). Large extracellular crystalline deposits in the stroma in Schnyder crystalline dystrophy (d). Guttae at the level of the endothelium in FuchsÕ endothelial dystrophy (e). Epithelioid cells with irregular size and shape, indistinct borders, and hyper-reßective nuclei in iridocorneal endothelial syndrome (f)

may assist clinicians not only in the diagnosis but also in their treatment decision. The list of epithelial and stromal dystrophies studied with IVCM includes epithelial basement membrane (EBMD), MeesmanÕs, Reis-BŸcklers, Thiel-Behnke, central mosaic, central cloudy dystrophy of Fran•ois, lattice, granular, Avellino (or granular-lattice), macular central dystrophy, and Schnyder dystrophies.

In MeesmanÕs corneal dystrophy, IVCM showed hypo-reßective areas corresponding to microcysts in the basal epithelial layer associated with large elongated epithelial clefts and round hyper-reßective structures probably corresponding to intracellular material (Fig. 4.3a) [6].

In EBMD patients, IVCM showed the presence of abnormal hyper-reßective linear structures or curved ridges within the intermediate and basal cell layers corresponding to an aberrant thickened basement membrane penetrating into the epithelium. This abnormal basement membrane was also associated with epithelial cell abnormalities and microcysts (Fig. 4.3b) [7].

In BowmanÕs layer dystrophies, IVCM demonstrated an abnormal reßective tissue replacing the normal bowmanÕs layer. Moreover, IVCM may be able to identify and to differentiate in vivo Thiel-Behnke and Reis-Bucklers corneal dystrophies [8].

In Reis-Bucklers dystrophy, a small granular hyper-reßective material was observed within the basal epithelium and the BowmanÕs layer (Fig. 4.3c). Distinctively, in Thiel-Behnke dystrophy, the deposits showed homogeneous reßectivity with round edges and dark shadows.

In Schnyder dystrophy, IVCM showed large extracellular crystalline deposits and a highly reßective extracellular matrix modify the corneal architecture with disruption of subepithelial nerve plexus (Fig. 4.3d).

In FuchsÕ endothelial dystrophy, using IVCM, guttae appear as round hyporeßective structures with sometimes a central reßective material at the level of the endothelium. These images are associated with a cellular polymegethism, a pleomorphism, and a decrease in endothelial cell density as evaluated quantitatively with IVCM (Fig. 4.3e). IVCM images of posterior polymorphous dystrophy showed curvilinear and vesicular abnormalities with focal clusters of dark and bright bodies associated with streaks and craters at the level of the Descemet membrane. In iridocorneal endothelial syndrome (ICE syndrome), IVCM images of the endothelium show epithelioid cells with irregular size and shape, indistinct borders, and hyperreßective nuclei (Fig. 4.3f).

Refractive Surgery

Refractive surgery is one of the areas where IVCM may be the most useful as it can be used to characterize in vivo cellular changes associated with the wound healing response, to evaluate different techniques, or to assess complications [9, 10]. The appearance of the epithelium, the corneal stroma, the interface, the keratocytes or the corneal nerves can be observed and analyzed over time and monitored noninvasively at high resolution.

After PRK, IVCM shows the regeneration of the epithelium that covers the wound. In the anterior stroma, keratocytes become hyper-reßective with visible cellular processes and with an increased density during the early post-operative period (Fig. 4.4a). Using IVCM, it has been observed that activated keratocytes were responsible for the clinically visible haze and interestingly, this corneal reaction could be evaluated quantitatively and objectively with this imaging technique [9]. Corneal nerves regeneration after PRK has also been evaluated using IVCM.

Numerous studies on LASIK have been conducted with IVCM. LASIK corneas exhibit microfolds at the level of BowmanÕs layer appearing as long dark lines with varying thickness and length, often vertically oriented. Observation of the ßap interface commonly demonstrates highly reßective particles of variable dimensions (Fig. 4.4b). However, the exact origin of these particles remains undetermined [11]. The density of keratocytes could be measured precisely with a decrease number of keratocytes in the initial wound healing phase on both sides of the cut, in the ßap and in the anterior cornea bed. A progressive loss of keratocytes in these areas was also observed 3 years after LASIK [12]. In the meantime other keratocytes below and above the ßap show signs of activation with reßective nuclei and visible cytoplasmic processes [11]. IVCM studies of corneal nerves after LASIK have shown that in the sub-basal and stromal ßap regions, the number of nerve Þber bundles

Fig. 4.4 In vivo confocal microscopy (IVCM) images (400 × 400 mm, Heidelberg Retina Tomograph Ð Rostock Cornea Module (HRT-RCM)) of the cornea after refractive surgery. Activated keratocytes are hyper-reßective with visible cellular processes (a). Flap interface with highly reßective particles of variable dimensions (b). Abnormal corneal nerves (c). Flap margin after IntraLase¨ ßap creation (d)

decreased by more than 90% 1 week after LASIK. Sub-basal and stromal ßap nerve Þber bundles gradually increased from month 3 up to 1 year postoperatively, but regeneration appears to remain incomplete for as long as 3 years postoperatively (Fig. 4.4c) [13].

Complications of refractive surgery have also been studied with IVCM [9]. In cases of corneal inÞltrate, IVCM could help to differentiate diffuse lamellar keratitis (DLK) with inÞltration of cells considered most likely to be mononuclear cells and granulocytes into the interface ßap, from infectious keratitis. Finally, IVCM is interesting to characterize and compare the wound healing response and the corneal changes in novel techniques or devices used for refractive surgery (Fig. 4.4) [11].