Revisiting ciliary muscle tendons and their connections with the trabecular meshwork by two photon excitation microscopic imaging

PURPOSE. To elucidate the anatomy of the trabecular meshwork (TM) and its connection to ciliary muscle (CM) tendons with two photon excitation microscopic (TPEM) imaging. METHODS. The human aqueous outflow pathway was imaged in an unfixed and nonembeded state by using an inverted TPEM. Laser (Ti:Sapphire) was tuned at 850 nm for emission. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through 425/30-nm and 525/45 emission filters, respectively. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three-dimensional data sets. RESULTS. Collagen and elastin structures of the TM were successfully visualized with TPEM. The TM and CM tendons were found to contain both collagen and elastin fibers. What appears to be juxtacanalicular tissue (JCT) was identified by its honeycomb-like appearance in AF images. Tracing CM tendons from their origins and to their insertions revealed that elastin fibers of CM tendons were connected to the elastin network within the trabecular lamellae. The CM tendons converged or diverged along their course, forming intricate networks with the TM. The CM tendon fiber density varied depending on its location within the aqueous outflow pathway with tendons near the JCT found to be the most dense, and in a fine-tooth comb arrangement. CONCLUSIONS. By using TPEM imaging, new details of the human aqueous outflow pathway were elucidated. This high-resolution imaging technique revealed the intricate interconnections between the TM and CM tendons.