  Purpose: The rate of conventional aqueous humor outflow is the highest
nasally. We hypothesized that this is reflected in regionally different outflow
structures and analyzed the entire limbus by high-resolution, full-thickness
ribbon-scanning confocal microscopy (RSCM). Methods: We perfused pig eyes by
anterior chamber cannulation with eight lectin-fluorophore conjugates, followed
by optical clearance with benzyl alcohol benzyl benzoate (BABB). RSCM and an
advanced analysis software (Imaris) were used to reconstruct a
three-dimensional, whole-specimen rendering of the perilimbal outflow
structures. We performed morphometric analyses of the outflow tract from the
level of the trabecular meshwork (TM) to the scleral vascular plexus (SVP).
Results: Except for pigmented structures, BABB cleared the entire eye.
Rhodamine-conjugated Glycine max agglutinin (soybean, SBA) labeled the outflow
tract evenly and retained fluorescence for months. RSCM produced terabyte-sized
files allowing for in silico dissection of outflow tract vessel at a high
resolution and in 3D. Networks of interconnected lumina were traced from the TM
to downstream drainage structures. The collector channel (CC) volumes were ten
times smaller than the receiving SVP vessels, the largest of which were in the
inferior limbus. Proximal CC diameters were up to four times the size of distal
diameters and more elliptical at their proximal ends. The largest CCs were
found in the superonasal and inferonasal quadrants where the highest outflow
occurs. Conclusions: RSCM of cleared eyes enabled high-resolution, volumetric
analysis of the outflow tract. The proximal structures had greater diameters
nasally while the SVP was larger in the inferior limbus.
