Two-photon imaging of the mouse eye.

Glaucoma is a heterogeneous group of diseases that, as a group, is the second leading cause of blindness worldwide.1,2 Current diagnostic techniques for glaucoma, such as visual field testing and retinal nerve fiber layer measurements, lack disease specificity and provide little additional information beyond that of the common funduscopic examination. Currently, high intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, although it is well known that many patients with glaucoma do not have elevated IOP.3 Furthermore, methods for measuring IOP are predisposed to inaccuracies because of operator error, corneal biomechanical differences, and other poorly understood variables.4 Finally, the measurement of IOP is merely a surrogate metric for the health state of the outflow system of the eye, and more direct structural and functional assessments of aqueous outflow would be desirable. It is believed that structural abnormalities in the aqueous outflow pathway, especially in the juxtacanalicular trabecular meshwork (TM), the inner wall of Schlemm's canal (SC), or both, may lead to elevated IOP and the development of glaucoma.5 Visualizing the TM region at high resolution would be of potential diagnostic value for the early detection of glaucoma and for following its progression in patients. Although currently available clinical imaging technologies such as optical coherence tomography (OCT) and ultrasound biomicroscopy (UBM) can image SC,6,7 they are unable to achieve high enough resolution to be of clinical value in most forms of glaucoma such as primary open angle glaucoma. Two-photon microscopy (2PM) is a nonlinear imaging technology with subcellular resolution capabilities that has been previously used to image ocular tissues without the need for fixation.8–11 2PM involves two infrared photons interacting simultaneously with a target molecule. When 2PM is used to excite endogenous fluorophores it is called two-photon excitation autofluorescence (2PAF), and it is similar to traditional confocal fluorescence microscopy except that fluorescence excitation results from the absorption of two photons instead of one. 2PM can also be used to image by a process called second harmonic generation (SHG). SHG occurs when non-centrosymmetric macromolecular structures (principally collagen) simultaneously “scatter” instead of absorb the two infrared photons. This scattered energy resolves as a single higher energy (blue) photon of exactly half the excitation wavelength. The use of 2PM is significantly different in several ways from conventional microscopy (see Refs. 9, 12 for reviews). The excitation source is a high-intensity near-infrared laser with extremely short pulse duration (10−15 seconds). The near-infrared photons have greater tissue penetration than the higher energy photons used in confocal microscopy. The two-photon interaction occurs only where the objective focuses the laser, offering resolution similar in many ways to that of confocal microscopy. However, the duration of the 2PM laser pulse is so short that thermal damage should not occur in the tissue under the general conditions used for imaging. Additionally, the two-photon laser used for imaging is distinct from the two-photon laser used for surgical photodisruption of the TM,13 which is 105 times more powerful. Our group previously demonstrated the use of 2PM to image deeply into the TM of flat-mounted human cadaver tissue while in its native, unfixed state.8 In the present study, we evaluated the usefulness of 2PM for imaging the TM and nearby outflow structures within intact enucleated unfixed and fixed mouse eyes. Our findings provide the foundation for pursuing live animal ophthalmic imaging to further characterize the in vivo structure and function of the aqueous outflow system.