Coverslip

For live imaging, pupae were mounted in halocarbon oil (series 700; Halocarbon Products) on a coverslip (Sigma) and imaged with a laser 510 scanning confocal microscope (ZEISS) at 25°C with a 40 × objective, using LSM Browser software (ZEISS). Z-stacks had a 3 μm step size. Pupal collection and staging were done as described by [27 ].

After the perfusion study, some of the anterior segments were placed in 4% paraformaldehyde (PFA) in PBS (Thermo Fisher Scientific) overnight at 4°C. The anterior segments were then cryoprotected in 30% sucrose (Thermo Fisher Scientific) dissolved in PBS for 1 day. Wedges, including the TM, were grossly cut under a surgical microscope from eyes that included baseline high-flow TM, baseline low-flow TM, residual high-flow TM after glue, and residual low-flow TM after glue. These tissue wedges were mounted in O.C.T. Compound (Thermo Fisher Scientific). Cryosections 10 µm thick (Leica Biosystems, Vista, CA, USA) were cut, mounted onto Superfrost Plus slides (VWR International, Radnor, PA, USA), dried, taken through ethanol steps (70%–100%) followed by xylenes, stained with hematoxylin and eosin (Epredia, Kalamazoo, MI, USA), and mounted with a coverslip (Sigma-Aldrich). Tissue-section images were captured using a digital slide scanner (NanoZoomer S360 Digital Slide Scanner, 0.5-µm/pixel resolution; Hamamatsu Phototonics, Bridgewater, NJ, USA).

Trisodium citrate (TC), gold (III) chloride trihydrate (HAuCl₄·3H₂O), ascorbic acid (AA), sodium borohydride (NaBH₄), cetyltrimethylammonium bromide (CTAB), rhodamine 6G (R6G), and bovine serum albumin (BSA) have been supplied by Sigma Aldrich (Italy) and used as received. Milli-Q water was used for the preparation of gold nanoparticles. LN, a rhombohedral crystal belonging to the point group 3 m, was bought from Crystal Technology Inc. in the form of both sides polished, 500 μm thick, z-cut, and 3-inch diameter. The wafer was cut into square samples 2 × 2 cm² in size by a standard diamond saw. The deposition slide was a coverslip, bought from Sigma Aldrich, consisting of an untreated glass substrate 24 × 60 mm in size and 0.2 mm thick.