"Anterior bead distribution as a function of size is conserved between live and perfused pig eyes. ( a ) Representative image of a yellow pellet (asterisk) and needle trace (arrow) after 4 h of perfusion indicates where two sizes of fluorescent <t>microbeads,</t> 20 nm (green) and 2000 nm (red), were deposited into the inferior vitreous in the dissected posterior eye cup ( n = 3). ( b ) Representative higher magnification stereomicroscope images suggested the 20 nm beads (green) dispersed more than the 2000 nm size (red), visible as a green corona around the pellet (arrows). ( c ) Outflow tissues in sections through the anterior segment were assessed in confocal projections following ITV microbead injections. Only the smaller 20 nm beads (green) were visible in the trabecular meshwork and angular aqueous plexus (asterisks), which was co-stained for CD31 (white, merge), while the larger 2000 nm beads (red) did not appear in this compartment ( n = 3, arrow indicates the location of the AC for orientation). Note that the orientation and location of the probed tissues are also indicated in the electronic supplementary material, figure S6. A corresponding z-stack side projection was also assessed to ensure beads remained within the sectioned tissue plane. ( d ) Anterior microbead distribution patterns were also observed 4 h following injections in live pig eyes showing similar 20 nm bead depositions in outflow tissues (green), but no 2000 nm beads (red) (n = 3). ( e ) Corresponding fluorescent bead numbers were measured from serial aqueous fluid samples from the same perfused eyes collected at 2 h and 4 h following ITV bead injection and normalized as a percentage of injected beads. A progressive time-dependent increase in 20 nm microbeads was observed, but 2000 nm microbeads remained near baseline ( n = 3, linear regression shown, note; error bars smaller than the data point do not plot). ( f ) Raw microbead numbers measured from aqueous fluid samples at 4 h from the live pig study were compared with 4 h aqueous fluid samples from perfused ex vivo pig eyes, indicating comparable ratios between the 20 nm and 2000 nm sizes (n = 4). There were dramatically higher concentrations of 20 nm beads than 2000 nm in both cases. However, there was a small but significant difference between live and perfused 2000 nm samples. In comparison, controls with no active perfusion (control) showed virtually no aqueous signal detected ( n = 4). (Scale bars indicate 50 µm, data are mean ± s.e.m., *** p < 0.005, * p < 0.05, n.s.; not significant). "