1：Experimental Design and Method Selection:

The study employed high-speed volumetric multiphoton microscopy (MPM) imaging and image processing to generate 3D maps of metabolism within full-thickness, excisional wounds of diabetic and non-diabetic mice over 10 days. Methods included in vivo MPM for autofluorescence imaging, fluorescence lifetime imaging (FLIM), and ex vivo tissue analysis with immunohistochemistry.

2：Sample Selection and Data Sources:

C57BL/6J mice (6 weeks; male) were used, divided into streptozotocin-induced diabetic and control groups. Wounds were created using a sterile biopsy punch, and imaging was performed at days 1, 3, 5, 7, and 10 post-wounding.

3：List of Experimental Equipment and Materials:

Bruker Ultima Investigator laser scanning microscope, Ti:sapphire laser (Spectra-Physics), 20x, 1.0 NA water-immersion objective (Olympus), GaAsP photomultiplier tubes (Hamamatsu), filters (Chroma), ReliOn Blood Glucose monitor, Tegaderm bandages, Tissue-Tek OCT compound, Ki67 antibody, DAPI stain, and other reagents.

4：0 NA water-immersion objective (Olympus), GaAsP photomultiplier tubes (Hamamatsu), filters (Chroma), ReliOn Blood Glucose monitor, Tegaderm bandages, Tissue-Tek OCT compound, Ki67 antibody, DAPI stain, and other reagents. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Mice were anesthetized and imaged with MPM at wound edges. Z-stacks were acquired rapidly to minimize motion artifacts. Image processing involved registration, averaging, and normalization. FLIM data were collected for NADH lifetime analysis. Ex vivo sections were prepared for H&E staining and Ki67 immunolabeling.

5：Data Analysis Methods:

Statistical analysis used two-factor ANOVAs with Tukey's HSD tests and Pearson correlation coefficients. Image processing and analysis were performed in MATLAB and SPCImage software.