Confocal microscopy versus two-photon microscopy: imaging of ocular surface pathologies

Minimal-invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without the necessity of actual tissue probing. For this matter confocal microscopy with the Cornea Module, mounted on a laser scanning microscope, is in daily use in ophthalmic practise. Two-photon microscopy is a new optical technique that enables high resolution imaging and functional analysis of living tissues based on tissue autofluorescence with minimal phototoxic damage. This study was set up to compare the potential of two-photon microscopy to the established Cornea Module. Different ocular surface pathologies such as pterygia, papillomae, nevi and cysts were investigated using the Cornea Module for confocal microscopy in-vivo. The pathologies were excised, stored in tissue culture media and immediately investigated by two-photon microscopy without further fixation. After imaging, the specimens were sent for definite histopathological assessment. Cornea Module and two-photon microscopy both generated high resolution images of the investigated tissues. At wavelengths of 710-730 nm two-photon microscopy exclusively revealed cellular structures whereas collagen fibrils were specifically demonstrated by second harmonic generation. Measurements of fluorescent lifetimes (FLIM) enabled the highly specific display of e. g. goblet cells or erythrocytes within capillaries. FLIM also enabled to demarcate nevus-cell clusters from epithelial cells. At the settings used, two-photon microscopy reaches higher resolutions than the Cornea Module and has the option of tissue specific signals by wavelengths tuning and fluorescence lifetime imaging which give additional information about the tissue. The Cornea Module allows intravital real-time imaging with less technical effort that leads to the visualization of dynamic processes such as blood flow. The parallel detection of two-photon excited autofluorescence together with confocal imaging could expand the possibilities of minimal-invasive investigation of the ocular surface towards functional analysis at higher resolutions.