Time-lapse and cleared imaging of mouse embryonic lung explants to study three-dimensional cell morphology and topology dynamics

Here, we present a protocol for collecting high-spatiotemporal-resolution datasets of undisturbed mouse embryonic epithelial rudiments using light-sheet fluorescence microscopy. We describe steps for rudiment dissection, clearing, and embedding for cleared and live imaging. We then detail procedures for light-sheet imaging followed by image processing and morphometric analysis. We provide protocol variations for imaging both growing and optically cleared lung explants to encourage the quantitative exploration of three-dimensional cell shapes, cell organization, and complex cell-cell dynamics. For complete details on the use and execution of this protocol, please refer to Gómez et al. (2021).1 [Display omitted] •Flexible protocol for imaging live and cleared mouse rudiments•Adaptation of tissue clearing protocol for mouse embryonic lung explants•Whole-mount immunostaining and embedding protocol for 3D microscopy•Light-sheet fluorescence live microscopy and morphometric analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for collecting high-spatiotemporal-resolution datasets of undisturbed mouse embryonic epithelial rudiments using light-sheet fluorescence microscopy. We describe steps for rudiment dissection, clearing, and embedding for cleared and live imaging. We then detail procedures for light-sheet imaging followed by image processing and morphometric analysis. We provide protocol variations for imaging both growing and optically cleared lung explants to encourage the quantitative exploration of three-dimensional cell shapes, cell organization, and complex cell-cell dynamics.