1：Experimental Design and Method Selection:

The study used a nonlinear microscopy system based on a femtosecond Cr:forsterite laser at 1260 nm to perform third-harmonic generation (THG) and three-photon fluorescence (3PF) imaging. The design rationale was to enable virtual sectioning without physical slicing, leveraging the nonlinear optical properties of H&E stains.

2：Sample Selection and Data Sources:

Human skin tissues, including normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and extramammary Paget's disease (EMPD) specimens, were obtained from surgical samples stored in refrigerated saline. Tissues were prepared as thin sections or whole-mount layers (approximately 1 mm thick) to simulate Mohs micrographic surgery conditions.

3：List of Experimental Equipment and Materials:

Key equipment included a Cr:forsterite laser, inverted microscope (Olympus IX71), objectives (e.g., UAPON 40XW340), spectrometers (Andor Shamrock 303i), PMTs (Hamamatsu R4220P, R928P), and various optical components (lenses, filters, beamsplitters). Materials included H&E dyes (hematoxylin from Sigma-Aldrich, eosin from J.T.Baker), formaldehyde, ammonia solution, and cover glasses.

4：Experimental Procedures and Operational Workflow:

Tissues were stained using a simplified H&E protocol (formaldehyde, hematoxylin, ammonia, eosin, washing steps). Nonlinear spectroscopy was performed to characterize dye emissions. Imaging involved focusing the laser beam on samples, collecting THG and 3PF signals epi-collected by the objective, separating signals with dichroic beamsplitters and filters, and detecting with PMTs. Images were acquired with 512x512 pixels, frame rate of

5：5 fps, and accumulated for quality. Z-stacking was used for depth imaging. Data Analysis Methods:

Spectral data were analyzed using Gaussian fitting and power dependency studies (cubic fitting). Images were processed in ImageJ for color remapping based on Beer-Lambert law with attenuation constants for H&E. Spatial resolution was measured using FWHM of point sources.