Fig. 1: The volumetric multimodality microscopy imaging system and the method for volumetric data acquisition and reconstruction.

a Schematic drawing of the volumetric multimodal microscopy optical imaging system based on vertical sectioning tissue imaging. A femtosecond (fs) laser beam after passing through a polarizing beamsplitter was scanned in the x-direction by a resonance scanning mirror and relayed to the back aperture of the objective. The focal point of the laser beam is scanned in the z-direction by moving the objective using a piezo positioner. Two-photon fluorescence (TPF) and second harmonic generation (SHG) signals are collected by the objective and reflected by the dichroic mirror and the beamsplitter to two photomultiplier tubes (PMTs). Reflectance confocal (RCM) signal is collected by the objective and reflected to the avalanche photodiode (APD) by the polarizing beamsplitter after passing through the dichroic mirror and the scanning mirrors. b The procedure for volumetric data acquisition and motion correction. Volumetric data was acquired by simultaneously scanning in the xz direction and (stretching) moving the skin with the translation stage in the y-direction. The acquired raw data is then reconstructed after skin surface detection, motion detection, motion correction, and frame averaging process (see details in the “Methods” section).