Towards the three-dimensional imaging of murine vaginal tissue
Pelvic organ prolapse (POP) is the descent of the pelvic organs. Quantifying composition in 3-D as opposed to traditional 2-D histologic analysis may reveal new information on vaginal microstructure to inform diagnostics and design criteria to improve interventions on POP. Dual-view inverted selective plane illumination microscopy (diSPIM) images a tissue in 3-D using fluorescent stains. The fluorescent stain TO-PRO-3 Iodide and Eosin (T&E) is a potential analog to Hematoxylin and Eosin (H&E) that can be used to acquire 3-D images. Therefore, the goal of this study was to validate T&E as an H&E fluorescent analog and to acquire 3-D images of the murine vagina using diSPIM with T&E staining to observe morphological changes throughout the vagina. Staining of murine samples with T&E and H&E generated very similar images, validating the use of T&E as a fluorescent analog to H&E. diSPIM imaging with T&E staining applied to murine vaginal samples at varying stages of pregnancy generated 3-D images from which 2-D slices could be isolated. These 2-D slices were analyzed by delineation and thickness measurements of their vaginal layers (epithelial, subepithelial, and muscularis layers). These thickness measurements showed changes in thicknesses across depths and across pregnancy status in the vagina, such as the thickening of the epithelial layer throughout pregnancy. These results highlighted the capability of diSPIM to reveal information on the vaginal microstructure throughout its depth.