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 vertebrate species⁴. In particular, zebrafish models have contributed substantially to understandingthe genetic and physiological basis of heart formation, remodeling, and function under both normal and pathological conditions^5,6.

Structurally, the zebrafish heart is composed of a single atrium and a single ventricle, connected in a linear fashion, lacking the septation typical of the mammalian four-chambered heart. Despite this, the zebrafish heart recapitulates key aspects of human cardiac physiology, including the presence of endocardial and myocardial layers, a cardiac conduction system, rhythmic contractile activity, and regulation by neurohormonal factors^7,8. Importantly, the zebrafish heart exhibits key morphogenetic events, including the rightward looping of the heart tube and the subsequent expansion and delineation of cardiac chambers, resembling those in human embryonic development⁹. Furthermore, the zebrafish heart maintains many of the electrophysiological and hemodynamic properties seen in higher vertebrates, including action potential propagation, contractility modulation, and calcium cycling dynamics⁷.

Given these similarities, zebrafish have emerged as a pivotal model in toxicology, especially for evaluating how chemical exposures affect heart development and function¹⁰. However, one of the major limitations in this field remains the reliance on technically demanding imaging techniques. Conventional high-resolution methods, such as confocal or light-sheet microscopy, or transgenic lines expressing fluorescent cardiac markers, while powerful, are costly and require significant technical infrastructure¹¹. These constraints limit the broader implementation of zebrafish-based cardiac phenotyping, particularly in settings with restricted resources or for applications requiring high-throughput screening¹².

There is, therefore, a growing need for methodological approaches that allow reliable assessment of cardiac function using widely available instrumentation. Existing literature does contain examples of such simplified methods, including brightfield or light microscopy-based imaging combined with manual or semi-automated measurements^13,14. However, these techniques are not always standardized and often vary between research groups in terms of protocol, timing, and image analysis parameters. This variability hinders reproducibility and limits comparative value across studies, particularly in fields such as embryotoxicology, pharmacology, or ecotoxicology, where quantitative cardiovascular endpoints are increasingly used as indicators of developmental safety¹⁵.

In this context, zebrafish offer a valuable model for screening developmental cardiotoxicity in vivo. Regulatory agencies and scientific consortia have increasingly recognized the relevance of zebrafish in this area, especially given their capacity to detect subtle changes in cardiac function that may precede structural malformations or lethality¹⁴. The transparent embryo allows direct visualization of the beating heart, enabling time-resolved, live assessment of cardiac performance without the need for invasive procedures. Moreover, the simplicity of the zebrafish cardiac anatomy facilitates quantification of atrial and ventricular dynamics, including chamber dimensions and stroke parameters, using basic imaging techniques¹⁵.

The overarching goal of the method described in this article is to provide an accessible, reproducible approach for the morphofunctional assessment of zebrafish embryonic cardiac performance using standard light microscopy. By focusing on simple video acquisition and post hoc image analysis, the technique aims to fill the methodological gap between high-resolution imaging and qualitative observational scoring. This protocol is adaptable to diverse laboratory settings and resource levels, and is especially applicable to toxicological, pharmacological, or genetic studies focused on cardiac function evaluation.

In addition to its practical advantages, the protocol contributes to the field by offering a consolidated, step-by-step workflow that combines commonly used techniques into a single, reproducible pipeline. While similar approaches exist in the literature, few provide this level of integration with visual documentation, which may support wider adoption and standardization.

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.
2. Feed fish three times daily with a high-protein commercial flake or granular feed suitable for zebrafish.
3. In the late afternoon, transfer adult zebrafish to breeding tanks equipped with a transparent mesh to separate fish from the tank bottom. Use a male-to-female ratio of 2:3, separated overnight by a removable divider.
4. On the following morning, remove the divider and add 50-100 mL of cold aquarium water to trigger spawning.
5. After fertilization (within 30-60 min), remove adult fish from the breeding tank to prevent predation of embryos.
6. Collect embryos using a plastic Pasteur pipette and transfer them to sterile Petri dishes filled with E3 embryo medium.
   NOTE: E3 embryo medium is a buffered salt solution that mimics the ionic composition of zebrafish extracellular fluid. Prepare E3 medium by dissolving 580 mg of NaCl, 27 mg of KCl, 97 mg of CaCl₂·2H₂O, and 163 mg of MgCl₂·6H₂O in approximately 800 mL of ultrapure distilled water. Adjust the pH to 7.2 using 1 N NaOH or HCl. Once the pH is adjusted and stable, bring the total volume to 1,000 mL with distilled water. Sterilize the solution by filtering it through a 0.22 µm membrane filter. Add 100 µL of 1% methylene blue per liter to prevent fungal growth. Store the prepared E3 medium at 4 °C and warm to room temperature before use.
7. Incubate embryos at 28.5 °C in an incubator with a controlled light/dark cycle (14 h/10 h).
   NOTE: Limit the number of embryos per Petri dish (e.g., 50 per 100 mm dish) to avoid overcrowding, which can cause hypoxia and developmental defects.
8. Replace the E3 medium daily to maintain optimal developmental conditions.

2. Selection of morphologically normal embryos

1. At approximately 6 h post-fertilization (hpf; time elapsed since fertilization), transfer the fertilized embryos into a clean Petri dish containing fresh E3 medium.
2. Examine embryos under a stereomicroscope at 40x magnification.
3. Discard any embryos showing signs of developmental abnormality, including:
   1. Coagulated embryos: Identify by their opaque or white appearance, granular texture, and absence of distinguishable internal structures. Coagulation is a sign of nonviability and typically occurs within the first few hours after fertilization.
   2. Delayed development: Exclude embryos that have not progressed to the expected morphological stage (e.g., lack of epiboly or irregular cellular arrangement), suggesting impaired viability.
   3. Yolk sac abnormalities: Look for ruptured, irregular, or asymmetrical yolk sacs that may indicate defective development or osmotic imbalance.
   4. External malformations: Exclude embryos with twisted body axes, irregular cell mass distribution, or abnormal shapes of the chorion.
   5. Poor chorion integrity: Refrain fromusing the embryos with collapsed, shriveled, or ruptured chorions, as they may be subject to mechanical or osmotic stress.
4. Retain only embryos that meet all of the following criteria: (1) Transparent and symmetrical body shape, (2) Uniform epiboly progression with smooth cellular layering, (3) Clearly visible blastoderm and intact spherical chorion.
   NOTE: At 6 hpf, a morphologically normal zebrafish embryo is dome-shaped with a clearly defined blastoderm enveloping the top of the yolk. The cytoplasm should be evenly distributed, without granular speckling, and without signs of fragmentation or clumping.

3. Cardiac imaging using light microscopy

1. At 96 hpf, mount embryos individually in 35 mm glass-bottomed Petri dishes filled with E3 medium.
2. Prepare 1.2% low-melting-point agarose in E3 medium and preheat to 37-38 °C. Maintain the temperature using a heat block or water bath.
3. Embed each embryo in agarose and gently orient it laterally using fine forceps before the agarose solidifies.
   ​NOTE: Ensure that the heart is clearly visible and facing laterally, with both the atrium and ventricle in the same focal plane.
4. Use an inverted light microscope equipped with a digital camera to record 30-s high-resolution videos focused specifically on the cardiac region of the zebrafish embryos. Capture videos at either 40× or 100× magnification to ensure clear visualization of cardiac structures and heartbeats.
   1. Record the videos at a frame rate of 30 frames per second (fps) in uncompressed or minimally compressed formats (e.g., AVI or MOV) to preserve image quality and allow accurate analysis of cardiac motion dynamics. This setup enables detailed assessment of functional parameters such as heart rate, contraction rhythm, and chamber morphology.

4. Image analysis and measurement of cardiac function

1. Manual measurements in ImageJ
   1. Import Videos. Open videos in ImageJ using File > Import > AVI. If needed, convert the video into an image stack via Image > Stacks > Convert Images to Stack.
   2. Calibrate the measurement scale. Use Analyze > Set Scale, input the known field diameter (e.g., 4 mm for a 10× objective), and enable the Global option to apply the scale across all measurements.
   3. Define Regions of Interest (ROI). Use the ROI tools (line, polygon, rectangle) to mark the long and short ventricular axes during end-systole and end-diastole (Figure 1). Manage ROIs via Analyze > Tools > ROI Manager.
2. Measure the diameters and areas for calculating functional parameters (Supplementary File 1)
   1. Determine the Ventricular surface area (using the ellipse approximation) using Equation 1:
      VA = ((D_L - D_S)/2) × π (1)
      ​Where DL = long diameter, DS = short diameter.
   2. Estimate the ventricular volume at ED and ES using the ellipsoid volume formula (Equation 2)
      ​VV = (1/6) × π × D_L × D_S² (2)
   3. Calculate fractional area change (FAC) and fractional shortening (FS) using Equation 3 and Equation 4:
      FAC (%) = ((VAD - VAS) / VAD) × 100 (3)
      FS (%) = ((D_L - D_S) / D_L) × 100 (4)
      ​Where VAD = ventricular area at ED; VAS = ventricular area at ES; DDL = long axis at ED; DSS = short axis at ES.
   4. Determine the stroke volume (SV) using Equation 5:
      SV = VVD - VVS (5)
   5. Calculate the ejection fraction (EF)using Equation 6:
      ​EF (%) = (SV / VVD) × 100 (6)
3. Automated analysis in ZembryoAnalyzer
   1. Launch and load the captured video. Open ZembryoAnalyzer (available at: https://github.com/darkopuflovic/ZembryoAnalyser), and use File > Open Video to import the desired video.
   2. Perform the automatic Heart Region detection. Click on the Detect Heart button to allow the software to automatically locate the region of interest (ROI) based on brightness fluctuations across video frames. Adjust ROI manually if needed using rectangle, ellipse, or polygon tools.
   3. Perform the automatic Heart Rate estimation. Let the software analyze brightness changes within the detected ROI. Heart rate is calculated by identifying periodic peaks in intensity. The algorithm applies threshold filtering to remove noise and false positives, ensuring accurate beat detection.
   4. Export the results. Export the heart rate data and the corresponding intensity plots using Export > PDF or Export > Excel. Optionally, save the raw data in CSV or JSON format for downstream statistical processing.
4. Calculate the cardiac output (CO) using Equation 7:
   CO = SV × HR (7)

5. Assessment of protocol applicability in cardiotoxicity testing using toxicants

1. Weigh cadmium chloride and prepare a working solution by dissolving it directly in E3 medium to a final concentration of 3 mg/L.
2. Transfer the embryos into Petri dishes containing the cadmium solution at 24 h post-fertilization (hpf).
3. Incubate the embryos in the cadmium solution until 96 hpf at 28 °C± 1 °C with a 14-h light/10-h dark cycle.
4. Replace the cadmium solution daily to maintain consistent exposure.
5. At 96 hpf, record high-resolution cardiac videos, as previously described, of the embryos for subsequent measurement and analysis of cardiac function parameters.
   NOTE: Use this cadmium exposure protocol to simulate developmental cardiac toxicity in zebrafish embryos, as described in a previous study¹⁶.

6. Data management and replicates

1. Record all measurements in structured spreadsheets. Include embryo ID, treatment group, and all calculated parameters.
2. Ensure at least 20-30 embryos per group are analyzed for statistical reliability.
   NOTE: Video recordings of the cardiac region can be stored and analyzed at a later time point without compromising the accuracy of morphometric or functional measurements. In our internal comparisons, results from immediate and delayed analyses were consistent across keycardiac parameters, supporting the reproducibility of delayed video-based assessment.

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