1：Experimental Design and Method Selection:

The study used spectral two-photon microscopy (TPM), including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), to image the AOS in intact mouse eyes. The design involved using long infrared excitation wavelengths (930 nm) to reduce scattering and background, with image stacks collected for 3D rendering and segmentation. Anatomical references were established using perfusion techniques with fluorescent dyes.

2：Sample Selection and Data Sources:

Experiments were performed on eyes from adult C57BL/6 mice (age 12–20 weeks) of either sex, enucleated immediately after euthanasia. Some experiments involved cardiac perfusion with FITC-dextran or trans-corneal perfusion with fluorescent beads.

3：List of Experimental Equipment and Materials:

Equipment included a Zeiss LSM 780 inverted confocal microscope, a Spectra-Physics Ti:Sapphire laser, a 34X/1.2NA water-immersion objective, a QUASAR detector, a Harvard Apparatus syringe pump, a Hamilton micro-syringe, and custom-built eye-holders. Materials included PBS, FITC-labeled dextran, fluorescent polystyrene beads, and Davidson's Fixative.

4：2NA water-immersion objective, a QUASAR detector, a Harvard Apparatus syringe pump, a Hamilton micro-syringe, and custom-built eye-holders. Materials included PBS, FITC-labeled dextran, fluorescent polystyrene beads, and Davidson's Fixative. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Eyes were placed on a custom holder, perfused if applicable, and imaged with TPM. Laser power was adjusted for depth compensation. Image stacks were acquired with specific parameters (e.g., pixel dwell time 1.54 μs). Post-processing involved segmentation and 3D rendering using software like ImageJ, Seg3D, and FluoRender.

5：54 μs). Post-processing involved segmentation and 3D rendering using software like ImageJ, Seg3D, and FluoRender. Data Analysis Methods:

5. Data Analysis Methods: Image analysis included brightness/contrast adjustments, segmentation based on intensity thresholds, and quantitative measurements of AOS structures using software tools. Statistical analysis involved calculating means and standard deviations from multiple measurements.