Functional Cardiac Imaging in Zebrafish Embryos Using Standard Microscopy and Video Analysis: Applications in Environmental and Biomedical Research

High-resolution microscopy, fluorescent reporters, and digital image analysis, are some technologies that enable detailed assessment of embryonic cardiac function in zebrafish. Maintaining consistent imaging conditions and minimizing embryo movement remain major challenges for accurate cardiac function measurements. To begin, record videos of zebrafish embryos at 96 hours post in uncompressed formats, to allow accurate analysis of cardiac motion dynamics.

Sequentially, click on File, Open, then choose the AVI file in ImageJ to import the videos. Use Analyze, then click on Set Scale, input the known field diameter, and enable the global option to apply the scale across all measurements. Use the ROI tools to mark the long and short ventricular axes during end-systole and end-diastole.

Then determine the ventricular surface area using the ellipse approximation formula. Estimate the ventricular volume at end-diastole and end-systole using the ellipsoid volume formula. Now calculate the fractional area change and fractional shortening.

Then determine the stroke volume, SV, and ejection fraction, EF.For automated analysis, launch the Z-Embryo Analyzer software and click on File, then open Video, to import the desired video. Click on the Detect Heart button to allow the software to automatically locate the region of interest based on brightness fluctuations across video frames. Let the software analyze brightness changes within the detected region of interest to estimate heart rate, then calculate cardiac output.

The graphs show ventricular chamber area and volume measured at end-diastole and end-systole in zebrafish embryos at 96 hours post fertilization. The quantitative differences between the end-diastole and end-systole reflect normal systolic function during early cardiac development, supporting the applicability of this method. In healthy zebrafish embryos, the average stroke volume was 0.213 nanoliters.

Cardiac output was 27.8 nanoliters per minute. Ejection fraction was 41.99%and the average heart rate was 130 beats per minute. In healthy zebrafish embryos, the average fractional area change was 61.2%and fractional shortening was 44.5%Cadmium-exposed embryos exhibited abnormal cardiac morphology with enlarged ventricles and pericardial edema.

In cadmium-exposed embryos, stroke volume was significantly higher than in controls. Cardiac output was increased, and heart rate was reduced. The protocol addresses the lack of standardized accessible methods for quantifying zebrafish embryonic cardiac function with reproducible workflows.

This protocol uses standard microscopy and open-source analysis, making high-quality cardiac assessment feasible without specialized equipment. The findings provide a reliable protocol that improves comparability between studies and enables more precise evaluation of cardiac phenotypes.