Name: Functional Cardiac Imaging in Zebrafish Embryos Using Standard Microscopy and Video Analysis: Applications in Environmental and Biomedical Research (Video) | JoVE Journal
Uploaded: 2025-10-10T12:40:00.000+00:00
Duration: 4 min 11 s
Description: Learn to assess cardiac function in zebrafish embryos using standard microscopy and open-source video analysis. This reproducible protocol enables developmental cardiotoxicity studies without advanced imaging systems.

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5 minutes

This work was funded by the Ministry of Science, Technological Development, and Innovation of the Republic of Serbia, through an institutional funding agreement with the University of Belgrade, Faculty of Medicine, for the implementation and financing of research activities in 2025. The contract registration number for institutional funding in 2025 is 451-03-137/2025-03/200110. We kindly request that this number be included in the acknowledgments of all publications arising from the approved subprojects.

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
<ol>
	<li>Maintain adult zebrafish (Danio rerio) in a recirculating aquarium system at 28 &#176;C&#177; 1 &#176;C under a 14 h light/10 h dark cycle.</li>
	<li>Feed fish three times daily with a high-protein commercial flake or granular feed suitable for zebrafish.</li>
	<li>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.</li>
	<li>On the following morning, remove the divider and add 50-100 mL of cold aquarium water to trigger spawning.</li>
	<li>After fertilization (within 30-60 min), remove adult fish from the breeding tank to prevent predation of embryos.</li>
	<li>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&#183;2H2O, and 163 mg of MgCl2&#183;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 &#181;m membrane filter. Add 100 &#181;L of 1% methylene blue per liter to prevent fungal growth. Store the prepared E3 medium at 4 &#176;C and warm to room temperature before use.</li>
	<li>Incubate embryos at 28.5 &#176;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.</li>
	<li>Replace the E3 medium daily to maintain optimal developmental conditions.</li>
</ol>
2. Selection of morphologically normal embryos
<ol>
	<li>At approximately 6 h post-fertilization (hpf; time elapsed since fertilization), transfer the fertilized embryos into a clean Petri dish containing fresh E3 medium.</li>
	<li>Examine embryos under a stereomicroscope at 40x magnification.</li>
	<li>Discard any embryos showing signs of developmental abnormality, including:
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Yolk sac abnormalities: Look for ruptured, irregular, or asymmetrical yolk sacs that may indicate defective development or osmotic imbalance.</li>
		<li>External malformations: Exclude embryos with twisted body axes, irregular cell mass distribution, or abnormal shapes of the chorion.</li>
		<li>Poor chorion integrity: Refrain fromusing the embryos with collapsed, shriveled, or ruptured chorions, as they may be subject to mechanical or osmotic stress.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3. Cardiac imaging using light microscopy
<ol>
	<li>At 96 hpf, mount embryos individually in 35 mm glass-bottomed Petri dishes filled with E3 medium.</li>
	<li>Prepare 1.2% low-melting-point agarose in E3 medium and preheat to 37-38 &#176;C. Maintain the temperature using a heat block or water bath.</li>
	<li>Embed each embryo in agarose and gently orient it laterally using fine forceps before the agarose solidifies. 
	&#8203;NOTE: Ensure that the heart is clearly visible and facing laterally, with both the atrium and ventricle in the same focal plane.</li>
	<li>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&#215; or 100&#215; magnification to ensure clear visualization of cardiac structures and heartbeats.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Image analysis and measurement of cardiac function
<ol>
	<li>Manual measurements in ImageJ
	<ol>
		<li>Import Videos. Open videos in ImageJ using File &#62; Import &#62; AVI. If needed, convert the video into an image stack via Image &#62; Stacks &#62; Convert Images to Stack.</li>
		<li>Calibrate the measurement scale. Use Analyze &#62; Set Scale, input the known field diameter (e.g., 4 mm for a 10&#215; objective), and enable the Global option to apply the scale across all measurements.</li>
		<li>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 &#62; Tools &#62; ROI Manager.</li>
	</ol>
	</li>
	<li>Measure the diameters and areas for calculating functional parameters (Supplementary File 1)
	<ol>
		<li>Determine the Ventricular surface area (using the ellipse approximation) using Equation 1: 
		VA = ((D_L - D_S)/2) &#215; &#960; (1) 
		&#8203;Where DL = long diameter, DS = short diameter.</li>
		<li>Estimate the ventricular volume at ED and ES using the ellipsoid volume formula (Equation 2) 
		&#8203;VV = (1/6) &#215; &#960; &#215; D_L &#215; D_S&#178; (2)</li>
		<li>Calculate fractional area change (FAC) and fractional shortening (FS) using Equation 3 and Equation 4: 
		FAC&#160;(%) = ((VAD - VAS) / VAD) &#215; 100 (3) 
		FS (%) = ((D_L - D_S) / D_L) &#215; 100 (4) 
		&#8203;Where VAD = ventricular area at ED; VAS = ventricular area at ES; DDL = long axis at ED; DSS = short axis at ES.</li>
		<li>Determine the stroke volume (SV) using Equation 5: 
		SV = VVD - VVS (5)</li>
		<li>Calculate the ejection fraction (EF)using Equation 6: 
		&#8203;EF (%) = (SV / VVD) &#215; 100 (6)</li>
	</ol>
	</li>
	<li>Automated analysis in ZembryoAnalyzer
	<ol>
		<li>Launch and load the captured video. Open ZembryoAnalyzer (available at: https://github.com/darkopuflovic/ZembryoAnalyser), and use File &#62; Open Video to import the desired video.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Export the results. Export the heart rate data and the corresponding intensity plots using Export &#62; PDF or Export &#62; Excel. Optionally, save the raw data in CSV or JSON format for downstream statistical processing.</li>
	</ol>
	</li>
	<li>Calculate the cardiac output (CO) using Equation 7: 
	CO = SV &#215; HR (7)</li>
</ol>
5. Assessment of protocol applicability in cardiotoxicity testing using toxicants
<ol>
	<li>Weigh cadmium chloride and prepare a working solution by dissolving it directly in E3 medium to a final concentration of 3 mg/L.</li>
	<li>Transfer the embryos into Petri dishes containing the cadmium solution at 24 h post-fertilization (hpf).</li>
	<li>Incubate the embryos in the cadmium solution until 96 hpf at 28 &#176;C&#177; 1 &#176;C with a 14-h light/10-h dark cycle.</li>
	<li>Replace the cadmium solution daily to maintain consistent exposure.</li>
	<li>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.</li>
</ol>
6. Data management and replicates
<ol>
	<li>Record all measurements in structured spreadsheets. Include embryo ID, treatment group, and all calculated parameters.</li>
	<li>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.</li>
</ol>

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<li>Schlaeppi, A., Graves, A., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+sheet+microscopy+of+fast+cardiac+dynamics+in+zebrafish+embryos%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Light sheet microscopy of fast cardiac dynamics in zebrafish embryos.</a> J Vis Exp. 174, e62741(2021).</li>
<li>Motta, E. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Development+of+microscopy+strategies+for+rapid+imaging+of+zebrafish+heart+development.+Doctoral%22">Development of microscopy strategies for rapid imaging of zebrafish heart development. Doctoral dissertation</a>. , Université de Strasbourg. https://theses.fr/2020STRAJ043 (2020).</li>
<li>Spomer, W., Pfriem, A., Alshut, R., Just, S., Pylatiuk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+screening+of+zebrafish+embryos+using+automated+heart+detection+and+imaging%5BTitle%5D)+AND+%22J+Lab+Autom%22%5BJournal%5D)">High-throughput screening of zebrafish embryos using automated heart detection and imaging.</a> J Lab Autom. 17 (6), 435-442 (2012).</li>
<li>Santoso, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+methods+for+cardiac+rhythm+detection+in+zebrafish%5BTitle%5D)+AND+%22Biomed%22%5BJournal%5D)">An overview of methods for cardiac rhythm detection in zebrafish.</a> Biomed. 8 (9), 329(2020).</li>
<li>Teixidó, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+morphological+feature+assessment+for+zebrafish+embryo+developmental+toxicity+screens%5BTitle%5D)+AND+%22Toxicol+Sci%22%5BJournal%5D)">Automated morphological feature assessment for zebrafish embryo developmental toxicity screens.</a> Toxicol Sci. 167 (2), 438-449 (2019).</li>
<li>Mitovic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cadmium+significantly+changes+major+morphometrical+points+and+cardiovascular+functional+parameters+during+early+development+of+zebrafish%5BTitle%5D)+AND+%22Environ+Toxicol.+Pharmacol%22%5BJournal%5D)">Cadmium significantly changes major morphometrical points and cardiovascular functional parameters during early development of zebrafish.</a> Environ Toxicol. Pharmacol. 87, 103723(2021).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+An+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fiji: An open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li>
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</ol>

The authors have nothing to disclose.

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...

<a href="https://app.jove.com/t/6661">This corrects</a> the article <a href="https://dx.doi.org/10.3791/68941">10.3791/68941</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/68941">Functional Cardiac Imaging in Zebrafish Embryos Using Standard Microscopy and Video Analysis: Applications in Environmental and Biomedical Research</a>. The Abstract and Representative Results sections were updated.

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

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 development1,2. 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 conditions5,6.
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 factors7,8. 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 measurements13,14. 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.

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

functional cardiac imaging in zebrafish embryos using standard microscopy and video analysis: applications in environmental and biomedical research

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

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&#160;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.

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 = ...

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.

Zebrafish (Danio rerio) are widely used as a vertebrate model in cardiovascular research due to their genetic similarity to humans, optical transparency ...

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.

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JoVE Journal

Developmental Biology

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Learn to assess cardiac function in zebrafish embryos using standard microscopy and open-source video analysis. This reproducible protocol enables developmental cardiotoxicity studies without advanced imaging systems.

Functional Cardiac Imaging in Zebrafish Embryos Using Standard Microscopy and Video Analysis: Applications in Environmental and Biomedical Research (Video) | JoVE Journal

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

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