1：Experimental Design and Method Selection:

Human corneas were imaged with an inverted two photon excitation fluorescence microscope. The excitation laser (Ti:Sapphire) was tuned to 850 nm. Backscatter signals of SHG were collected through a 425/30-nm bandpass emission filter. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three dimensional data sets. ImageJ software was used to analyze the arrangement pattern (irregularity) of collagen bundles at each image plane.

2：Sample Selection and Data Sources:

Seven eye bank corneas, which were not suitable for human transplantation were obtained from the Central Florida Lions Eye and Tissue Bank (Tampa, FL, USA).

3：List of Experimental Equipment and Materials:

Inverted TPEF microscope (FluoView FV-1000; Olympus, Central Valley, PA, USA), Ti:Sapphire laser, 425/30-nm bandpass emission filter, glass-bottom dish (35 mm; MatTek, Ashland, MA, USA), balanced salt solution (BSS; Alcon, Fort Worth, TX, USA).

4：Experimental Procedures and Operational Workflow:

The cornea was placed on the glass-bottom plate, a laser was tuned to 850 nm and directed through a dichroic mirror, and a water immersion objective was used to focus the excitation beam and to collect backscatter signals; the SHG signal was collected through a bandpass emission filter after reflection by a dichroic mirror, a square image was acquired with 1024 x 1024 pixels of resolution in approximately 15 seconds, and multiple, consecutive, and overlapping image stacks (z-stacks) were acquired using the same objective lens.

5：Data Analysis Methods:

ImageJ software was used to analyze acquired images. Grayscale SHG images were processed by sequential application of image modifications including spatial frequency filters and binary conversion.