Use of confocal microscopy to determine matrix and surface protein deposition profiles in hydrogel contact lenses

We present a new method that combines protein-specific fluorescence staining with confocal microscopy to simultaneously measure matrix and surface protein deposits via optical sectioning of hydrogel contact lenses. Tetramethylrhodamine isothiocyanate (TRITC), Texas Red, fluorescein isothiocyanate (FITC), and fluorescein succinate were used to selectively react with the lysine residues of protein deposits for two lens types (etafilcon A [a 58% water, ionic lens] and polymacon [a 38% water, nonionic lens]). Because TRITC and Texas Red gave high levels of nonspecific staining, they were discontinued. Following exhaustive rinsing of lenses, central lens buttons were analyzed using a Bio-Rad MRC-500 laser scanning confocal microscope. Optical sections were made every 4 microns at 200x magnification, and the fluorescence signals were processed using image analysis software. The high water ionic material accumulated protein much more rapidly than the low water nonionic material. For a given lens type a correlation was observed between the wear time and the degree of protein deposition; however, we did observe significant inter-subject variations in total protein. From this preliminary work, we conclude that this confocal method is feasible and desirable for simultaneous determination of surface and matrix protein deposition.