Light sheet fluorescence microscopy pipelines for imaging and analysis of the human placental vasculature across gestation.

Over the first trimester, vasculogenesis (the de novo formation of blood vessels) and angiogenesis (creation of new vessels from existing vessels) together create the backbone of the placental vascular network, which becomes increasingly complex as gestation progresses to term. Many different ex vivo imaging modalities, including computed tomography, synchrotron X-ray tomography, confocal and electron microscopy, and traditional histology and stereology, have been used to study the vascularity of the human placenta in detail. However, many of these techniques often come with trade-offs between resolution, maximum sample size and cost. Light sheet fluorescence microscopy is an attractive method capable of imaging placental 3D morphology at the micro-scale, whilst also enabling imaging of comparatively larger placental explants than other microscopy techniques, allowing capture of more generations of villous and vascular trees. Advances in tissue clearing for optical imaging have led to a multitude of different published light sheet fluorescence microscopy protocols across tissue types. However, optimising placenta-specific protocols is imperative for high-quality imaging and downstream three-dimensional analysis of this unique tissue. Finally, the placental vascular architecture is notoriously disorganised, heterogenous and convoluted, making it more challenging to segment and obtain meaningful data than from vasculature in other organ systems. Here, we present a comprehensive pipeline for placental tissue preparation, light sheet fluorescence imaging and image segmentation of first trimester and term human placental villous explants, enabling physiologically relevant quantitative analysis of vascularity at different stages of gestation.