Background

The heart begins to beat early during morphogenesis [1]. While most studies on heart development have been performed in fixed tissue samples or in vitro, the development of the heart is regulated by the heartbeat and blood flow [2], and a full understanding of heart development requires performing studies in the beating heart.

Thanks to its transparency, the zebrafish embryo provides an excellent model to study heart development in vivo [3]. Using the Tg(fli1a:galff);(uas:GCaMP7a) zebrafish line, we recently demonstrated that mechanoresponsive calcium signals in valve endothelial cells are present from the earliest stages of valve formation and persist until functional valve leaflets are formed [4]. Calcium signal analysis in single z-slice movies of the beating heart is challenged by the way cells move both within and outside the image plane during heartbeat and the fact that the heart rate can change over the course of imaging. Since there was no readily available software to analyse calcium signals in the beating heart, we developed our own. This protocol provides step-by-step instructions on how to use the software, which can be used to further study calcium signalling in the developing heart or easily adapted for the analysis of other imaging data containing recurring events.

Our image analysis method involves the use of two MATLAB applications developed in our lab as well as FIJI [5], an open-source image processing package based on ImageJ2. The Phase Matching MATLAB application utilises the structural similarity score [6] to select frames corresponding to a particular phase in the cardiac cycle. The output of the Phase Matching application is a phase-matched movie with minimal cardiac cell movement. The Calcium Analysis MATLAB application reads the phase-matched movie and measures the normalised calcium signal intensity over time in a user-defined region of interest (ROI). FIJI is used to manipulate image stacks and to combine animated graphs of the normalised calcium signal with the phase-matched image data.