Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy

Abstract

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as postimplantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.

Forces and mechanotransduction in 3D vascular biology

“…Adapted from [6]. C) (top) Timelapse images of sprouting angiogenesis (white arrows) and anastomosis events (blue arrows) in E8.5 yolk sacs from [64]. (bottom) Timelapse images of flow-driven vessel arterial fusion events in yolk sacs from [47].…”

The Regulatory Landscape of Lineage Differentiation in a Metazoan Embryo

“…3D, time-lapse imaging now allows in toto imaging of metazoan embryogenesis in different model organisms and tracking of individual cells (Bao et al, 2006; Keller et al, 2008; McMahon et al, 2008; Udan et al, 2014; Wu et al, 2013; Xiong et al, 2013). In C. elegans , it allows direct tracing of the whole cell lineage (Bao et al, 2006; Santella et al, 2014; Santella et al, 2010).…”

Live Imaging of Mouse Secondary Palate Fusion

“…The best method for cell tracking depends on the reporter signal. If a nuclear reporter mouse is used, Imaris can trace the center of fluorescent signals as cellular centers 21 . Nuclear GFP reporter mouse such as ROSA26 GFP-NLS-lacZ ( GNZ) 22 can therefore be used to label epithelial cells using tissue-specific Cre lines; in our hands, this specific nuclear reporter exhibited faster bleaching over the long time-course used in live imaging.…”