In toto imaging of embryogenesis with confocal time-lapse microscopy

Abstract

Microscopy has been one of the most direct and powerful tools since the beginning of biological research. Continued advances such as confocal and two-photon fluorescence microscopy and fluorescent proteins now make imaging useful at a variety of spatial scales (molecules, circuits, cells, tissues, and even whole embryos) and temporal scales (<seconds to days). Zebrafish is uniquely poised to benefit from these continued technological improvements because of its inherent suitability for both imaging and genetics. This chapter presents an approach called "in toto imaging". The goal of in toto imaging is to image and track every single cell movement and division that forms a tissue or organ. This approach is powerful for understanding how cell lineage, shape changes, and movements control the morphogenesis of a tissue. When used with transgenic lines, in toto imaging can be used to "digitize" data at single cell level over time from a living organism. This quantitative, digitized data can then serve as the basis for forming models of how biological circuits orchestrate developmental processes.

Figure 1

Design for a lateral embryo array template

Figure 2

Design for a dorsal embryo array template.

Figure 3

Correct initial position for shield stage embryos in lateral mounts. The vegetal pole should be pointing ~20 degrees above the horizontal and ~20 degrees out of the page

Figure 4

A single xy image extracted from a very large in toto image to show a typical image. This image is a dorsal-lateral view of the otic vesicle and hindbrain. Nuclei are marked in green with H2B-EGFP and cell membranes are marked in red with a membrane localized mCherry.