Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools

Abstract

This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3-6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis.

Advances in Imaging Plant Cell Dynamics

“…The above setup is supported by the currently existing LSFM platforms, as it allows very fast optical sectioning and rapid image acquisition. Therefore, the total effective excitation energy absorbed by the sample in LSFM can be several orders of magnitude lower in comparison with the epifluorescence wide-field or confocal laser scanning systems (Ichikawa et al, 2014;Stelzer, 2015). The sample mounted on a rotor can be rotated during multiangular acquisition, allowing precise postacquisition rendering for 4D (x, y, z, and t) image reconstruction.…”

Preparation of plants for developmental and cellular imaging by light-sheet microscopy

“…Wide-field epifluorescence illumination will excite nearly all fluorophores in the sample producing blurred images that require postacquisition deconvolution 18 . Two-photon microscopy permits illumination of a very narrow optical plane; however, such systems are expensive and slow 19 .…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…As examples of specimens being imaged with high quality using light sheet microscopy, we should point out invertebrates such as D. melanogaster [48], C. elegans [58], and T. castaneum [59], and vertebrates such as zebrafish [47,60] (for very useful information regarding zebrafish preparation for laser sheet imaging see [61]). Of particular importance is the work by Ichikawa et al where light sheet microscopy was applied to a developing mouse embryo, given the complexity of keeping a mouse embryo under controlled CO 2 and temperature conditions [62]. In this work an approach for transferring embryos to the laser sheet microscope without exposing them to the environment is presented.…”