Figure 1

Figure 1

Workflow of automated imaging and heart rate quantification in medaka and zebrafish embryos. (a) Cardiovascular development is functional after 24 and 48 hpf and hatching occurs around 48–72 hpf and 7–8 dpf in zebrafish and medaka, accordingly, defining time windows of embryonic cardiac imaging in both fish species. (b) Single or multiple fish embryos per well are mounted without anaesthesia or agarose into microtiter plates, which have a fixed position in the imaging platform; image sequences can be sampled at different frame rates, resolutions and channels; all images were recorded with 2x objective, images with single embryos were cropped, multiple embryos in a well (Multi) are presented as full frame; in the fluorescent image A and V denote atrium and ventricle and a rough outline of the embryo is overlaid as a reference. (c) After image optimization, image sequences are quantified through a GUI of the HeartBeat program (compare Fig. 2) and heart rates, beat-to-beat variability and an overview of segmentation are saved as spreadsheet and graphical output, respectively.