Two-Photon Imaging of the Mouse Eye

PURPOSE. To image the conventional aqueous outflow pathway and adjacent structures within the intact enucleated mouse eye using a noninvasive microscopy technique. METHODS. Two-photon microscopy (2PM) techniques, including two-photon autofluorescence (2PAF) and second harmonic generation (SHG), were used to obtain images of the trabecular meshwork (TM) region within an intact mouse eye. Cardiac perfusion of fluorescein-conjugated dextran was used to label blood vessels within the eye to serve as an anatomic reference. Eyes were subsequently fixed, paraffin embedded, sectioned, and stained for comparison to the 2PM images. RESULTS. Three-dimensional analyses of multiple 2PM images revealed a well-defined region adjacent to the iris and cornea that is free of SHG signal and consistent with the location of Schlemm's canal. This open region is continuous with smaller tube structures consistent with collector channels. These structures do not label in mice perfused with the vascular probe dextran, supporting the hypothesis that the enclosed spaces are filled with aqueous humor rather than circulating blood. The TM region in the mouse eye was also visible, with a clear SHG signal representing collagen fibers. CONCLUSIONS. These results support the hypothesis that 2PM may be useful for noninvasively imaging the conventional aqueous outflow pathway in mouse models of glaucoma. Studies are ongoing to validate our methodology in live animals. (Invest Ophthalmol Vis Sci. 2011; 52:4098-4105) DOI:10.1167/iovs.10-7115