3D visualization of uterus and ovary: tissue clearing techniques and biomedical applications

Abstract

Recent advancements in tissue clearing and three-dimensional (3D) visualization technologies have enabled subcellular-level examination of entire organs, particularly in complex structures such as the ovary and uterus. Traditional histological approaches are limited by two-dimensional views, which restrict our understanding of female reproductive system functions. In this review, we highlight the innovations in 3D tissue clearing techniques applied to uterine and ovarian tissues, which, combined with analytical tools, facilitate comprehensive 3D visualization and image analysis. We evaluate the advantages and disadvantages of three primary categories of tissue clearing techniques: organic solvent-based, hydrogel-based, and hydrogel-embedded methods, specifically regarding the uterus and ovary. Light-sheet and multiphoton microscopy complement these techniques, providing unprecedented capabilities for high-resolution imaging of large tissue volumes. Tissue clearing technologies provide a robust strategy for early diagnosis of uterine and ovarian pathologies. Additionally, we explore the integration of tissue clearing technologies with spatial transcriptomics and AI-driven analytical tools to achieve comprehensive 3D molecular mapping. We hope this review contributes to a better understanding of tissue clearing techniques and can help researchers in navigating methodological choices for uterine and ovarian investigations.

Keywords: 3D visualization; AI; ovary; spatial omics; tissue clearing; uterus.

FIGURE 1

Major tissue clearing methods are applied to ovarian and uterus tissues. Created with BioRender.com.

FIGURE 2

Decision trees for choosing clearing protocols on the basis of the integrity, size and fluorescence signals of samples. N, no; Y, yes; IF, immunofluorescence; EF, endogenous fluorescence; W, weak; S, strong. Created with BioRender.com.

FIGURE 3

Principles of confocal, multiphoton, and light-sheet microscopy. (A) Confocal microscopy: a pinhole filters the illuminant to a point source focused by the objective lens. This enables optical sectioning via point-scanning in the x-y plane. Deeper scanning captures sequential images, and computer processing constructs the 3D structure. (B) Multiphoton microscopy: nonlinear excitation enables deeper tissue penetration. Illustrated with epi-fluorescence detection using one photomultiplier tube (PMT). (C) Light-sheet microscopy: a cylindrical lens forms a static light sheet illuminating the sample plane along the z-axis. Parallel illumination across the field of view minimizes photodamage. Detection occurs orthogonally (x-y plane). Created with BioRender.com.