Chameleon ultra 2 laser

Posterior scleral specimens were mounted in a custom inflation chamber, such that the posterior surface of the LC was aligned with the objective of a Zeiss 710 laser-scanning microscope (LSM 710 NLO; Zeiss, Inc., Oberkochen, Germany). Specimens were kept hydrated with 1M phosphate-buffered saline throughout the inflation test. Pressure was applied by using a water column. Each specimen was pressurized first to a baseline of 5 mm Hg then to 45 mm Hg. At both pressures, the specimen was allowed to equilibrate for at least 30 minutes before two sets of 2 × 2 tiled z-stacks were acquired, starting from a z-depth of 300 μm below the posterior surface and moved toward the posterior surface with a z-increment of 3 μm, using a Chameleon Ultra II laser tuned to 780 nm (Coherent Inc., Santa Clara, CA, USA), a 390- to 410-nm band pass filter to isolate the SHG signal, and a 10× 0.45 NA Apochromat objective. The zoom factor was varied between 0.6 to 0.8 depending on the size of the LC, which corresponded to an in-plane resolution of 2.08 to 2.77 μm/pixel. The tiles were imaged at 512 × 512 pixels and stitched with 15% overlap. The resulting 947 × 947-pixel images were exported as TIFF files (Fig. 1A).