Whole-mount sections displaying microvascular and glandular structures in human uterus using multiphoton excitation...

There has been considerable interest over many years in the precise structural relationships between microvessels and secretory glands in human endometrium. However, microcirculatory networks have rarely been studied in three-dimensions (3D) using modern computerised technologies, this has been partly due to the late arrival of suitable endothelial cell markers. This study was designed to develop a technique to visualize and to reveal the relationships between microvessels, their glandular environment and epithelial boundaries in 3D, using endometrium from human hysterectomy biopsies. Specimens were carefully selected from women with conditions unlikely to affect the microvascular networks. Monoclonal antibodies (mouse anti-human CD 34 and goat anti-mouse fluorescein (FITC)) were used to visualize the microvessels, and polyclonal antibodies (rabbit anti-human keratin and goat anti-rabbit tetramethylrhodamine (TRITC)) were used to visualize the glandular structures. The samples were studied with a Leica multiphoton system using a titanium-sapphire laser (excitation 800 nm with pulses in the 200 fs range) to obtain a stack of two-dimensional (2D) images to a minimal focus depth of 120 microm. The initial data sets acquired were volume rendered using the integrated software of the Leica system to produce 3D images. This software allowed for the acquisition of data sets from the microscope and for an observational morphological assessment to be made, but was limited in preparing the data for any quantitative analysis. The additional use of ImarisBasic 3.1 visualization software allowed for an observational morphological assessment but also included numerous tools for data manipulation.