The procedure for SHGM imaging of fresh ex vivo retina is described in the following. The excitation for SHGM was 100-fs pulses from a Ti:Sapphire laser at 785-nm wavelength with 80-MHz repetition rate. A Pockels cell was employed to attenuate the laser beam and also to rapidly switch it off during the galvanometer’s return travel. The laser beam was focused with a water immersion objective lens (Olympus XLUMPlanFL ) on an upright microscope. The theoretical full width at half maximum of the squared excitation intensity is 0.28 and 1.1 μm in the transverse and axial dimension, respectively. The average power was approximately 50 mW at the sample. SHG was acquired in the forward detection path, which consists of a high-NA objective lens (Olympus UApo340 ), bandpass filters, and a PMT detector (Hamamatsu). The retinal flat mounts from adult female Sprague-Dawley rats were prepared as follows and approved by Hunter College Institutional Animal Care and Use Committee. Briefly, animals were euthanized and the eyes enucleated. A circumferential incision was made around the limbus, and the cornea, lens, and vitreous humor were removed and radial cuts made in the eye cup. The retina was separated from the retinal pigment epithelium and sclera and transferred to buffered Ames’ Medium bubbled with 95% and 5% at 34°C and pH 7.4. The sample was transferred to a perfusion chamber with a transparent bottom within 15 min after euthanasia. The retina was placed on a microscope stage with the RNFL side up. In order to investigate the relationship between the morphology of the retinal nerve fiber bundle and the MT network, we performed time-lapse SHGM. We induced dissociation of MTs in the RNFL using a chemical agent that hinders polymerization of the MTs (nocodazole, Sigma-Aldrich). Initially the fresh retina received only Ames’ Medium, then perfusion was switched at time zero to the second medium containing 25-µM nocodazole and 0.4% dimethyl sulfoxide (DMSO, Sigma-Aldrich).