Purpose: Elevated intraocular pressure due to dysfunction of the aqueous outflow system (AOS) is a major risk factor for developing open angle glaucoma. We propose to use multiphoton spectral imaging techniques to quantify the structural and functional changes of the AOS to better understand the development and progression of the disease.

Methods: Multiphoton spectral microscopy techniques, including two-photon autofluorescence (TPAF) and second harmonic generation (SHG), were used to image the structures of the AOS in freshly enucleated mouse eyes. To optimize optical penetration through the highly scattering scleral tissue, the excitation laser was tuned to longer infrared wavelengths (≥ 930nm) without appreciable signal degradation. SHG was used to identify the scleral and corneal collagen structures. In separate experiments, TPAF was used to identify blood vessels perfused with fluorescent-conjugated dextran (150KDa) or AOS perfused with fluorescein.

Results: An interconnected system of anterior chamber structures including Schlemm’s canal (SC), collector channels (CC) and perilimbal superficial vessels were identified using the absence of SHG signal from the scleral collagen. The collagen of the trabecular meshwork (TM) was detected proximal to SC. The diameter of the SC at the TM was ~61 µm and tapered down to ~11 µm where it connected to CC. The average depth of the SC was ~17 µm. The diameters of the CC and perilimbal superficial vessels were ~18.5 µm and ~15.3 µm respectively. None of these structures labelled with fluorescent dextran in cardiac perfusion experiments, indicating that they were not episcleral veins.

Conclusions: Detailed structures of the mouse TM, SC, CC, perilimbal superficial vessels and episcleral veins were identified and quantified using TPAF and SHG. These results validate the use of longer excitation wavelength (≥930nm) for deeper penetration of intact ocular tissues in multiphoton microscopy.