Confocal Laser-scanning Microscopy - Approach towards the In-Vivo Cytology of the Eye?

Stave J., Guthoff R.,
Universität Rostock, Augenklinik (Rostock)

Background: Owing to the development of confocal in-vivo microscopy on the basis of the confocal slit-scanning microscopy, besides specular microscopy of the endothelium also in-vivo imaging of optical cuts became possible in the micrometer range of the cornea and the conjunctiva bulbi. Confocal slit scanning microscopy, however, is system-dependently limited concerning dissolution and picture illumination. In refractive surgical procedures into the corneal structures, performed with a high-energy UV EXCIMER laser, the achievement of picture-morphologic parameters of the microarchitecture of the cornea before and after surgery with high local resolution and even picture illumination plays an increasing role. In combination with automatic evaluation systems, a computer-aided quantitative "cytology" becomes possible. Furthermore, the examination of the epithelium of the central and peripheral cornea as well as the adjacent conjunctiva is particularly interesting in connection with wound healing, reinnervation after surgery and carrying contact lenses. The aim was the development of a confocal laser scanning microscope for in-vivo investigations of high dissolution and photographic speed as well as image field illumination on the basis of a modern, digital and commercial laser-scanning system, which meets the requirements mentioned.
Material and method: We developed an z-scan adapter to the objective of the Heidelberg Retina Tomographe I/II, by the means of which, a misalignment of the laser focus takes place into the anterior eye segment. Thus, a high-solution, digitalconfocal laser-scanning microscope RLSM (Rostock Laser Scanning Microscope) was developed. Due to the application of an external z-scan device at the objective in connection with a contact element, an accurate depth evaluation of the epithelium, the nerves, the keratocytes and the endothelium can be performed.
Results: As the RLSM can be applied with a high picture-scanning speed in the millisecond range and a dissolution of lines within the micrometer range, a distortion-free imaging of the corneal structure can be performed in the entire image field with up to 600fold enlargement. For the first time confocal in-vivo microscopy in combination with a contact element can be used for accurate depth allocation < 1 mm. The epithelium, the nerval plexus, the keratocytes of the entire cornea, the endothelium as well as cell and tissue structures of the conjunctiva bulbi can be imaged in good quality. For the first time, an in-vivo histology became possible in the sense of a "cytology". Due to the application of the confocal laser scanning technology, the image quality has improved decisively in comparison to the slit-scanning technology.

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