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

Summary

This protocol describes a low-cost light microscopy method to assess cardiac morphology and function in zebrafish embryos, enabling reproducible evaluation of developmental cardiotoxicity without the need for advanced imaging systems.

Abstract

Zebrafish (Danio rerio) are widely used as a vertebrate model in cardiovascular research due to their genetic similarity to humans, optical transparency during early development, and amenability to in vivo imaging. This manuscript presents a standardized, accessible protocol for assessing cardiac morphology and function in zebrafish embryos at 96 h post-fertilization (hpf) using brightfield light microscopy. The method includes embryo collection, morphological screening, immobilization in agarose, video recording of the beating heart, and image-based analysis of cardiac parameters such as ventricular dimensions, stroke volume, heart rate, ejection fraction, and cardiac output. Calculations are based on geometric approximations of the ventricle using open-source software (e.g., ImageJ, Zembryo Analyzer). The protocol enables quantitative assessment of cardiac performance using widely available equipment, making it suitable for laboratories with limited resources and high-throughput screenings. In this study, 96 hpf zebrafish larvae were analyzed by recording the beating heart under a light microscope. Key cardiac parameters measured included ventricular dimensions, which were used to calculate stroke volume and heart rate, determined by beats per minute. From these, ejection fraction and cardiac output were derived to assess overall cardiac performance. Representative results from healthy embryos demonstrated consistent ventricular contraction and robust functional indices, while embryos exposed to cadmium showed impaired cardiac morphology and significantly increased cardiac output. Overall, the method is important for detecting developmental cardiotoxicity and provides a reproducible, non-invasive approach for evaluating cardiac function in vivo during early zebrafish development.

Introduction

Zebrafish (Danio rerio) have emerged as a prominent vertebrate model organism in cardiovascular research due to their genetic homology with humans, external fertilization, optical transparency of embryos, and rapid development^1,2. These features, combined with cost-effectiveness and scalability, make zebrafish particularly suitable for the study of early cardiac morphogenesis, congenital heart defects, and developmental cardiotoxicity³. Their relevance is further reinforced by the high conservation of genes and signaling pathways that regulate heart development across vertebrat....

Protocol

All procedures involving zebrafish embryos were conducted in accordance with the institutional animal care and use guidelines approved by the local ethics committee. Zebrafish husbandry and embryo manipulation were performed in compliance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Zebrafish maintenance and embryo collection

1. Maintain adult zebrafish (Danio rerio) in a recirculating aquarium system at 28 °C± 1 °C under a 14 h light/10 h dark cycle....

Results

This protocol allows for quantitative analysis of zebrafish cardiac development at 96 h post-fertilization (hpf), including structural and functional parameters such as ventricle size, contractility, stroke volume, and ejection fraction.

Figure 2 shows representative brightfield images of the zebrafish ventricle at end-diastole (ED) and end-systole (ES), along with corresponding bar graphs displaying average ventricular area (VA) and volume (VV) measurements (n = .......

Discussion

One of the important steps in this protocol is the accurate identification and selection of morphologically normal embryos at 6 h post-fertilization (hpf). Embryos that exhibit signs of coagulation, developmental delay, or structural anomalies can significantly compromise the consistency and interpretability of cardiovascular measurements. The early removal of nonviable or malformed embryos ensures a reliable baseline for assessing heart development and function. Proper orientation of embryos during imaging, ensuring lat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm Membrane Filter	Millipore	SLGP033RS	0.22 µm Membrane Filter
1 N HCl solution	Sigma-Aldrich	320331	Hydrochloric acid solution for pH adjustment
1 N NaOH solution	Sigma-Aldrich	72068	Sodium hydroxide solution for pH adjustment
Adult zebrafish (Danio rerio)	Local breeding facility	N/A	Model organism
Breeding tanks with mesh	/	/	Separation of adults for spawning
CaCl?·2H?O	Sigma-Aldrich	C5670	Calcium chloride dihydrate, reagent grade
Cadmium chloride	Sigma-Aldrich	202908	Toxicant used for exposure at 3 mg/L
Commercial zebrafish feed	Mini Gran, Nutri Pet, Gornji Milanovac	/	High-protein flake or granular feed suitable for zebrafish
Digital Camera (microscope)	Hamamatsu Photonics	ORCA-Flash4.0 V3	Camera for video recording at 30 fps
Heat block	Eppendorf	5382000010	Temperature controlled heating device
ImageJ Software	NIH	https://imagej.nih.gov/ij/	Open-source image processing software
Inverted Light Microscope	Nikon	Ti2-E	Microscope with digital camera, 40× and 100× objectives
KCl	Sigma-Aldrich	P3911	Potassium chloride, reagent grade
Low-melting point agarose (1.2%)	Thermo Fisher Scientific	16520050	For embedding embryos
Methylene Blue (1%)	Sigma-Aldrich	M9140	Antifungal agent added to E3 medium
MgCl?·6H?O	Sigma-Aldrich	M9272	Magnesium chloride hexahydrate, reagent grade
NaCl	Sigma-Aldrich	S9888	Sodium chloride, reagent grade
Sterile Petri Dishes (100 mm)	Corning	430167	Plastic Petri dishes for embryo culture
Sterile Petri Dishes (35 mm)	MatTek Corporation	P35G-1.5-14-C	Glass-bottom Petri dishes for mounting embryos
Ultrapure distilled water	Millipore	Milli-Q Reference	Water for solution preparation
ZembryoAnalyzer Software	GitHub (Darko Puflovic)	https://github.com/darkopuflovic/ZembryoAnalyser	Cardiac function analysis software