Like for any new imaging technique, analysis of the images is only possible after a thorough grounding in the semiology. The goal of this chapter is to present a large variety of IVCM and OCT applications in the cornea and anterior segment in order to render this new means of imaging more familiar, making it possible to use these very promising imaging techniques with optimum efÞciency.

In Vivo Confocal Microscopy (IVCM)

Despite its use over the last 20 years, it has not been until recently, with new commercially available IVCM devices, that IVCM has become more accessible to clinicians. Combined with high-performance digital imaging, IVCM offers today a non-invasive method of examining the cornea, the limbus or the conjunctiva with possible magniÞcation of up to 400 times.

Principles of Confocal Microscopy

The principle of confocal microscopy was described for the Þrst time by Marvin Minsky back in 1955 while studying brain parenchyma cells. He proposed that the observation (objective) and illumination (condenser) systems be focused in a single point, hence the name ÒconfocalÓ microscopy. By focusing the observation and illumination system on a single point, light reßected by elements outside the focal point could be excluded, thus increasing image resolution and contrast. The ability of in vivo confocal microscopy to eliminate the light out-of-focus allows up to a few microns axial and lateral resolution. However, the spatial resolution is improved at the cost of a considerable reduction in the Þeld of view (focal point). It is therefore necessary to quickly observe adjacent points and to reconstruct the images in order to allow direct observation of the whole specimen. Different techniques are used to rapidly capture all the points composing the image of the specimen. The Tandem Scanning Confocal Microscope (TSCM) contains a rotating Nipkow disc that has pinholes arranged in Archimedian spirals. This type of in vivo confocal microscope is no longer in production. The Slit Scanning Confocal Microscope (SSCM) uses two optically conjugate scanning optical slits for illumination and detection. SSCM are commercially available from Tomey Corporation (Cambridge, MA, USA), Nidek Technologies (Gamagori, Japan), and Helmut Hund (Wetzlar, Germany). The most recent development in clinical confocal microscopy is the Confocal Laser Scanning Microscope (CLSM) (Heidelberg Retina Tomograph Rostock Cornea Module (HRT-RCM), Heidelberg Engineering, Heidelberg, Germany). Compared to the TSCM and the SSCM that use a white light source, this recently developed confocal microscope uses a 670-nm red wavelength diode laser as a light source. In order to scan the whole specimen, the laser beam is scanned sequentially over each point of the examined area by a set of galvanometer scanning mirrors.

Fig. 4.1 In vivo confocal microscopy (IVCM) images (400 × 400 mm, Heidelberg Retina Tomograph Ð Rostock Cornea Module (HRT-RCM)) of the normal cornea. SuperÞcial epithelial cells (a). Basal epithelial cells (b). Bowman layer with sub-basal nerves (c). Anterior stroma with hyper-reßective keratocyte nuclei (d). Stromal nerve (e). Endothelium (f)

The Normal Cornea

With the exception of the normal Descemet membrane, the confocal microscope can provide images of all layers of the cornea. Contrary to conventional microscopy, which provides transverse sections of the tissue, images obtained with IVCM generally consist of optical sections oriented parallel to the surface observed.

The corneal epithelium consists of superÞcial, intermediate, and basal epithelial cells. SuperÞcial cells appeared as polygonal Ð often hexagonal Ð cells with a size up to 50 mm in diameter. The reßectivity is variable but cells undergoing desquamation are characterized by a highly reßective cytoplasm with a visible nuclei and a perinuclear dark halo (Fig. 4.1a). Intermediate epithelial cells or wing cells have a regular form with size up to approximately 20 mm, and are characterized by reßecting cell borders and a dark cytoplasm with rarely visible nuclei. The basal epithelial layer forms a regular mosaic of smaller cells (8Ð10 mm), with a dark cytoplasm, reßective cell borders, and without visible nuclei (Fig. 4.1b).

The sub-basal nerve plexus is located between BowmanÕs membrane and the basal epithelium. Sub-basal corneal nerves appear as hyper-reßective, beaded linear structures of 4Ð8 mm thickness (Fig. 4.1c). BowmanÕs membrane appears as an amorphous layer of 8Ð10 mm thickness located between basal cells and the stroma. Only the highly reßective stromal nerves and keratocyte nuclei are visualized on