Frontiers in heart development34Fast Digital Laser Sheet Microscopy: swimaging in 6 dimensions

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Zebrafish are increasingly used to investigate vertebrate heart formation. Hundreds of embryos that will develop in a predictable rapid sequence can be obtained cheaply and on demand. Hearts are easily visualised in these transparent embryos using fluorescent transgenic lines that can label cardiomyocytes, endothelial cells or other tissues types. As their cardiac development is conserved with mammals until septation, the sequenced genome and the ability to knock down or mutate specific genes have made them a powerful resource. Our laboratory has worked for several years in analysing how disruption of planar cell polarity (non-canonical Wnt) signalling disrupts cardiac development. These studies have been hampered by difficulties in obtaining enough embryos for a thorough analysis at the earliest stages of heart development and the problems of keeping mouse embryos alive during imaging. For these reasons, we have turned to zebrafish embryos and explored new imaging techniques.

Fast Digital Laser Sheet Microscopy (FastDLSM) permits three-dimensional visualisation of developing zebrafish embryonic hearts throughout the cardiac cycle (4th-dimension) and throughout development (5th-dimension). Simultaneous visualisation of multiple transgenic reporters determines a 6th dimension. We have developed and built a microscope system that projects a thin sheet of multiple wavelength laser light progressively through a live zebrafish embryo. High-speed digital cameras acquire images of the optical sections throughout the cycle and at different depths through the embryo. The image loops are registered within the cardiac cycle allowing a three dimensional movie to be constructed. The imaging chamber is heated and oxygen tension and pH can also be controlled.

Here we explain how the FastDSLM is used in the analysis of cardiac developmental processes with regard to both form and function. We show how our current investigations into planar cell polarity signalling and heart malformation in the mouse have been advanced using zebrafish and FastDLSM. Preliminary data showing how this technique is being applied to embryonic mouse heart development will also be presented.

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