We express our gratitude to Dr. Caroline Burns at Boston Children&#39;s Hospital for generously sharing the transgenic zebrafish. We thank Ms. Elizabeth Ibanez for her help in husbanding zebrafish at UT Dallas. We also appreciate all the constructive comments provided by D-incubator members at UT Dallas. This work was supported by NIH R00HL148493 (Y.D.), R01HL162635 (Y.D.), and UT Dallas STARS program (Y.D.).

Approval for this study was granted by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas at Dallas, under protocol number #20-07. Tg(myl7:nucGFP) transgenic zebrafish larvae12 were used for the present study. All data acquisition and image post-processing were carried out using open-source software or platforms with research or educational licenses. The resources are available from the authors upon reasonable request.
1. Z...

Analysis of Zebrafish Cardiac Contraction with Virtual Reality Interaction

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The integration of the zebrafish model with engineering methods holds immense potential for the in vivo exploration of myocardial infarction, arrhythmias, and congenital heart defects. Leveraging its optical transparency, regenerative capacity, and genetic and physiological similarities to humans, zebrafish embryos and larvae have become extensively utilized in research1,2,4. The superior spatiotemporal resolution, mini...

The zebrafish (Danio rerio) is a widely used model organism for studying cardiac development, physiology, and repair due to its optical transparency, genetic tractability, and regenerative capacity1,2,3,4. Upon myocardial infarction, while structural and functional changes impact the cardiac ejection and hemodynamics, technical limitations continue to hinder the ability to investigate the dynamic process during cardiac regeneration with the high spatiotemporal resolution. For example, conventional imaging methods, such as confocal microscopy, have limitations in terms of imaging depth, temporal resolution, or phototoxicity for capturing the dynamic changes and assessing cardiac contractile function during multiple cardiac cycles5.
Light-sheet microscopy represents a state-of-the-art imaging method that successfully addresses these issues by quickly sweeping the laser across the heart&#39;s ventricle and atrium, achieving detailed images with enhanced spatiotemporal resolution and negligible photo-bleaching and photo-toxic effects6,7,8,9,10,11.
This protocol introduces a comprehensive imaging strategy that includes LSM system construction, 4D image reconstruction, 3D cell tracking, and interactive analysis to capture and analyze the dynamics of cardiomyocytes across the entire heart during multiple cardiac cycles12. The customized imaging system and computational methodology allow&#160;one to track the myocardial microstructure and contractile function at the single-cell level in transgenic Tg(myl7:nucGFP) zebrafish larvae. Furthermore, small molecule compounds were delivered into the embryos using microinjection to assess drug-induced cardiac injury and subsequent regeneration. This holistic strategy provides an entry point to in vivo investigate structural, functional, and mechanical properties of myocardium at the single-cell level during cardiac development and regeneration.

<table>
	<tbody>
		<tr>
			<td>RESOURCE</td>
			<td>SOURCE/Reference</td>
			<td>IDENTIFIER</td>
		</tr>
		<tr>
			<td>Animal models</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(myl7:nucGFP) transgenic zebrafish</td>
			<td>Burns Lab in Boston Children&#39;s Hospital</td>
			<td>ZDB-TGCONSTRCT-070117-49</td>
		</tr>
		<tr>
			<td>Software and algorithms</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The MathWorks Inc.</td>
			<td>R2023a</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National Instruments Corporation</td>
			<td>2017 SP1</td>
		</tr>
		<tr>
			<td>HCImage Live</td>
			<td>Hamamatsu Photonics</td>
			<td>4.6.1.2</td>
		</tr>
		<tr>
			<td>Python</td>
			<td>The Python Software Foundation</td>
			<td>3.9.0</td>
		</tr>
		<tr>
			<td>Fiji-ImageJ</td>
			<td>Schneider et al.18</td>
			<td>1.54f</td>
		</tr>
		<tr>
			<td>3DeeCellTracker</td>
			<td>Chentao Wen et al.15</td>
			<td>v0.5.2</td>
		</tr>
		<tr>
			<td>Unity</td>
			<td>Unity Software Inc.</td>
			<td>2020.3.2f1</td>
		</tr>
		<tr>
			<td>Amira</td>
			<td>Thermo Fisher Scientific</td>
			<td>2021.2</td>
		</tr>
		<tr>
			<td>3D Slicer</td>
			<td>Andriy Fedorov et al.17</td>
			<td>5.2.1</td>
		</tr>
		<tr>
			<td>ITK SNAP</td>
			<td>Paul A Yushkevich et al.16</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Light-sheet system</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cylindrical lens</td>
			<td>Thorlabs</td>
			<td>ACY254-050-A</td>
		</tr>
		<tr>
			<td>4X Illumination objective</td>
			<td>Nikon</td>
			<td>MRH00045</td>
		</tr>
		<tr>
			<td>20X Detection objective</td>
			<td>Olympus</td>
			<td>1-U2M585</td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Hamamatsu</td>
			<td>C13440-20CU</td>
		</tr>
		<tr>
			<td>Motorized XYZ stage</td>
			<td>Thorlabs</td>
			<td>PT3/M-Z8</td>
		</tr>
		<tr>
			<td>Two-axis tilt stage</td>
			<td>Thorlabs</td>
			<td>GN2/M</td>
		</tr>
		<tr>
			<td>Rotation stepper motor</td>
			<td>Pololu</td>
			<td>1474</td>
		</tr>
		<tr>
			<td>Fluorescent beads</td>
			<td>Spherotech</td>
			<td>FP-0556-2</td>
		</tr>
		<tr>
			<td>473nm DPSS Laser</td>
			<td>Laserglow</td>
			<td>R471003GX</td>
		</tr>
		<tr>
			<td>532nm DPSS laser</td>
			<td>Laserglow</td>
			<td>R531003FX</td>
		</tr>
		<tr>
			<td>Microinjector and vacuum pump</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>WPI</td>
			<td>PV850</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Welch</td>
			<td>2522B-01</td>
		</tr>
		<tr>
			<td>Pre-Pulled Glass Pipettes</td>
			<td>WPI</td>
			<td>TIP10LT</td>
		</tr>
		<tr>
			<td>Capillary tip for gel loading</td>
			<td>Bio-Rad</td>
			<td>2239912</td>
		</tr>
		<tr>
			<td>Virtual reality hardware</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VR headset</td>
			<td>Meta</td>
			<td>Quest 2</td>
		</tr>
		<tr>
			<td>30mg/L PTU solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PTU</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
		</tr>
		<tr>
			<td>1X E3 working solution</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1% Agarose</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Low-melt agarose</td>
			<td>Thermo Fisher</td>
			<td>16520050</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>10g/L Tricaine stock solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Syndel</td>
			<td>SYNC-M-GR-US02</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
		</tr>
		<tr>
			<td>150mg/L Tricaine working solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10g/L Tricaine stock solution</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>60X E3 stock solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Lab Animal Resource Center (LARC), The University of Texas at Dallas</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>-</td>
			<td>KCL</td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>-</td>
			<td>CaCL2 x 2H2O</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>-</td>
			<td>MgSO4 x 7H2O</td>
		</tr>
		<tr>
			<td>RO Water</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>1X E3 working solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60X E3 stock solution</td>
			<td>Lab Animal Resource Center (LARC), The University of Texas at Dallas</td>
			<td>-</td>
		</tr>
		<tr>
			<td>RO Water</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1% Methylene Blue (optional)&#160;</td>
			<td>-</td>
			<td>C16H18ClN3S</td>
		</tr>
	</tbody>
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4d light-sheet imaging of zebrafish cardiac contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Our research uses 4D light-sheet imaging to explore cardiac contractile function in zebrafish, aiming to understand the dynamics of cardiac contraction at the single-cell level. One of the foremost experimental challenges is achieving high spatial terminal resolution, dynamic imaging of cardiac function while observing natural heart rhythms. Our study fills the gap in analyzing intricate 4D cardiac dynamic at single-cell resolution.We utilize GPU-based parallel computation, virtual reality, and advance cell tracking to interpret light-sheet imaging data. Our novel virtual reality platform offers an interactive approach to assess regional cardiac contractions, providing manipulative functionalities for more in-depth analysis. By facilitating user-directed analysis of cardiac contraction, our protocol could lead to an enhanced understanding of cardiac development and disease.

The current protocol consists of three main steps: zebrafish preparation and microinjection, light-sheet imaging and 4D image reconstruction, and cell tracking and VR interaction. Adult zebrafish were allowed to mate, the fertilized eggs were collected, and performed microinjection as needed for the proposed experiments (Figure 1). This step provides an entry point to explore zebrafish applications in the investigation of cardiac development and regeneration, and it also plays a crucial role...

Watch this Scientific Journal Video about 4D Light-sheet Imaging of Zebrafish Cardiac Contraction at JoVE.com

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

4d-light-sheet-imaging-of-zebrafish-cardiac-contraction