Accept a pair of globe from NDRI (day n).

• Receive the delivery at 10 am or 2 pm (day n+1).

• Make sure the eyes are well hydrated. If not, add BSS to the jar.

• Test the right eye (OD) on day n+1 and the left (OS) on day n+2. Clean extra orbital fat and muscle, cut the optic nerve flush with the sclera. Store the nerve in PFA for grading.

• Mark the nasal side of the sclera. The nasal/temporal axis of the eye is marked with a clear vein. The main muscle, which wraps around the sclera attaches on the temporal side of the ONH (See [24] for illustration).

• Measure the three main dimensions of the eye (NT, IS, AP), and pick the holder best fitting the eye.

• Cover the holder with glue and place it 2 mm posterior to the equator, with the ONH centered. Maintain a firm contact between the holder and the eye for at least 5 min- utes. To prevent rotation of the eye when pressing the holder onto the sclera, place the globe on a cornea holder. This will prevent the eye from rotating and will help centering the ONH.

• After the glue has dried (∼5-10minutes), cut the anterior segment of the eye, leaving a band of at least 5 mm of sclera below the holder.

• Remove the lens, iris, vitreous humor, choroid and retina, which should detach easily (if needed, store the retina in PFA). At this point, the sclera should be clean. The inner and outer surface should be white. Removing the choroid is more complicated for the bovine or porcine sclera.

• Score the band of the sclera left on the anterior side every 5 mm and impregnate the entire band with glue. Fold back the anterior scleral band onto the holder and add glue to secure the seal. I usually used at least half a tube of cyanoacrylate (Krazy Glue) per eye. Wait at least 15 mm or until the glue turns white, while maintaining the sclera moist by repeated applications of BSS.

At that point, the sclera should look as presented in Chapter 4, Fig. 4.1, and is ready to be placed on the inflation chamber. To ensure that the pressure remains within physiological, it is recommended to follow these steps.

Before starting, drain the water and tare the pressure on TestWork. Make sure the pressure chamber and the pressure transducer are stabilized and cannot rotate during the time of testing.

• With the chamber opened, fill the syringe with BSS and let the BSS flow until all air bubbles disappear in the line.

• Fill the pressure chamber with the syringe pump open and the valve closed.

• Screwthespecimenontothechambersuperiorpoleup,whilemonitoringthepressure in the chamber. It is necessary to release pressure during this process as it can reach 45-60 mmHg.

• Fill the plunger to the top. Without leak, the pressure should stabilize around 0.5 psi. If there is a leak, the pressure will slowly drop.

• Close the system by lowering the plunger. Keep an eye on the pressure in the cham- ber. As the plunger hits the water, the pressure will rise very quickly, so it is necessary to release the pressure to keep it below 30 mmHg.

• Pressurize to 0.28 psi to perform thickness measurements with the pachymeter. Plan to spend at least 30 minutes on this. I recommend to measure thickness at 8 locations on 3 circles (peripapillary, midposterior, outer sclera). Remember to keep the sclera hydrated. Only report thickness measurements when the pachymeter was able to measure quickly 50 times the same thickness.

• Airbrush the sclera with India Ink, or use graphite powder through the 62 μ m mesh. Note that applying India Ink is irreversible. The graphite powder can be removed.

• Bring the pressure to 0.03 psi or 1.5 mmHg by slowly opening the valve. Let the specimen equilibrate for at least 25 minutes at the baseline pressure. During that time, the pressure in the chamber will progressively increase. Regularly open the valve to bring the pressure back to 0.03 psi. Start the test only when the pressure can be maintained at 0.03 psi for a long period of time (at least 5 minutes).

• Create a folder on the Shuttle computer for the test and open VicSnap in this folder. Make sure that VicSnap is able to read the pressure from the other computer (DELL) If not, restart the Shuttle.

• Calibrate the cameras. The cameras should be focussed on the peripapillary sclera. The aperture should be 8 mm, and the stereo angle should be at least 15◦. Make sure that the fields of view of the 2 cameras overlap over the entire specimen. Switch off the light and take at least 50 pictures for the calibration. Rotate the calibration grid clockwise (20 times) and counter clockwise (20 times) and translate it in the plane of the sclera (10 times). Open Vic3D and run the calibration. This should give a calibration number lower than 0.03. Redo if it is larger than that. I recommend to have the correlation done before starting the test to verify the quality of the speckle pattern. Adjust the speckle pattern and the lighting if there are regions where Vic3D cannot calculate the displacements (glare, poor speckling pattern).

• Enclose the specimen in the humidity chamber and start the test. I recommend tak- ing a picture every second for the load-unload tests and every 2 seconds for the creep tests. I have had synchronization problem when taking more than one picture a second.

This part has to be done after testing to calculate the DIC displacements, usually overnight. I have noticed that capturing pictures with VicSnap and correlating the data with Vic3D at the same time can slow down the computer and affect the synchronization between the two computers. Load the calibration file and the test pictures into a Vic3D document, define the area of interest and start correlating. If the calibration was checked before testing, the correlation should work fine.