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M. Mueller, G. Huettmann, N. Koop, P. Steven; Minimal-Invasive Imaging of Ocular Surface Pathologies - Confocal vs. Two-Photon Microscopy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2258.
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Minimal-invasive imaging of ocular surface pathologies may give beneficial information prior to surgical procedures. We here compare already established confocal laser scanning microscopy with two-photon microscopy, a new optical method for minimal-invasive high resolution imaging.
Patients presenting ocular surface pathologies such as pterygium, pannus, conjunctival cysts, papilloma, nevus and pingueculum were examined with the Rostock Cornea Module (Heidelberg Engineering, Germany) prior to surgery. Immediately after the procedure the excised pathologies were examined by two-photon microscopy (DermaInspect, Jenlab, Germany) without previous fixation and tissue processing. Images were generated by exciting autofluorescence at different wavelengths. Fluorescence-lifetime imaging (FLIM) and Second Harmonic Generation (SHG) were conducted additionally. After two-photon microscopy, excised pathologies were fixed and processed for histopathological examination.
High resolution reflection images, generated by laser scanning confocal microscopy, demonstrate tissue components as epithelial cells, connective tissue fibres and deposits, blood vessels and erythrocytes. Two-photon microscopy of corresponding tissue structures allows to estimate the nucleoplasmic index, to identify macrophages and to distinguish cellular from acellular components by wavelength modulation and Second Harmonic Generation. Fluorescing-lifetime imaging (FLIM) allows to differentiate goblet cells and nevus cells from epithelial cells of the conjunctiva.
This is the first study to compare confocal with two-photon microscopy regarding ocular surface pathologies. Laser scanning confocal microscopy features advantages such as rapid image formation and real-time imaging of large tissue areas whereas two-photon microscopy enables additional characterization of individual cell types and tissue components by wavelength adjustment, FLIM and SHG. Possible applications could comprehend a general preoperative tissue examination by confocal microscopy and an additional two-photon examination of selected tissue components for in-detail analysis of pathological changes.
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