1：Experimental Design and Method Selection:

The study uses OR-PAM for high-resolution imaging of tumor microvasculature in mice. A novel algorithm involving preprocessing (Gaussian and high-frequency emphasis filtering), enhancement (Hessian matrix and intensity transformation), vascular segmentation (region growing), skeleton extraction (improved two-step multistencils fast marching method), and quantitative evaluation (calculating vessel diameter, microvascular density, vascular tortuosity, and fractal dimension) is employed.

2：Sample Selection and Data Sources:

In vivo experiments were conducted on mice with implanted melanomas and hepatomas. Phantom experiments used tungsten wires and black ink to mimic melanoma structures. Data were acquired from mouse ears over multiple days post-tumor implantation.

3：List of Experimental Equipment and Materials:

OR-PAM system components include a tunable diode-pumped laser (NT242-1K-SCU EKSPLA), a 75-MHz ultrasonic transducer (V2022, Olympus-NDT), an electrical amplifier (ZFL-500LN-BNC+, Mini-Circuits), and a data acquisition card (CS1422, GaGe). Mice (BALB/c and Balb/c nude), tumor cells (B16 melanoma and Hep G2), and anesthesia (isoflurane) were used.

4：Experimental Procedures and Operational Workflow:

Mice were anesthetized and imaged using OR-PAM with laser wavelengths of 570nm and 1064nm. Images were processed through the algorithm steps to extract and quantify vascular features. Phantom experiments validated the system's capability.

5：Data Analysis Methods:

Quantitative parameters were computed from extracted skeletons using MATLAB functions and custom algorithms for statistical analysis, including box-dimension method for fractal dimension and various metrics for tortuosity.