Our protocol presents an optimized tool to study the zebrafish heart in vivo and describes embedding immobilization techniques along with the data acquisition and analysis pipeline for cardiac imaging. Zebrafish is a cardiac model system with a large potential for applications like drug screen. Protocol that enable gentle imaging under physiological conditions are crucial when studying heart diseases.
The workflow presented here focuses on zebrafish embryonic heart imaging, but a modified version can be applied to various other samples and experiments, including squid, worm, and hydra. Begin by collecting the embryos obtained from the breeding of the desired transgenic lines. Keep the embryos at 28 degrees Celsius in a Petri dish filled with E3 fish medium.
Use a bore glass needle mounted onto a micromanipulator and connected to a pico injector to inject 30 picograms of alpha-bungarotoxin mRNA into the yolk of one or two cell stage embryos to immobilize the fish. Keep the eggs at 28 degrees Celsius in a Petri dish filled with E3 medium, and transfer the eggs every 24 hours to a new dish containing fresh E3 medium until imaging. To prevent pigment formation, if the zebrafish background is not albino, transfer fish at 25 hours post fertilization to a new dish containing E3 medium with 0.2 millimole tyrosinase inhibitor 1-phenyl 2-thiourea, also known as PTU.
To straighten the fluorinated ethylene propylene tube place the tube in a glass or steel autoclave safe tubing with the correct inner diameter to fit the polymer tubes and autoclave at 180 degrees Celsius for two hours. Once the tubes reach room temperature, remove the straightened polymer tube. For cleaning the tube, use a 50 millimeter syringe to flush the tube twice with one molar of sodium hydroxide.
Cut the tube to the size of a 50 milliliter centrifuge tube, place in a centrifuge tube filled with 0.5 molar of sodium hydroxide and ultrasonicate for 10 minutes. Flush the fluorinated ethylene propylene tube with double distilled water, followed by 70%ethanol. Transfer tubes a fresh centrifuge tube contained 70%ethanol and ultrasonicate for 10 minutes.
After ultrasonication, flush the tube for the last time with double distilled water, and store it in a centrifuge tube containing double distilled water. After dissolving low melting point agarose in E3 medium pour the melted agarose in a glass or plastic Petri dish to make a one to two millimeter coat. Once the agarose is solidified, gently pour the E3 medium on top of the agar to prevent drying.
Seal the plate with paraffin film and store at four degrees Celsius. Thaw the stock solution of tricaine and add 0.02%tricaine to the E3 medium without methyl blue to use as embedding medium. Use a disposable glass pipette to transfer fish to the embedding medium.
Verify that the fish has stopped moving and the heart is beating at a similar speed compared to the control. Cut the fluorinated ethylene propylene tube to the ideal length with a razor blade. Prepare a syringe with a blunt end cannula.
Fill the syringe with air, then mount the tube onto the needle and gently flush out any remaining water by emptying the syringe. Next, fill the fluorinated ethylene propylene tube mounted on a syringe with media. Insert an embryo in the tube, keeping the fish head close the tube end, avoiding bubble formation.
Using a razor blade, carefully cut the tube at the edge of the blunt end cannula, or needle. After discarding any liquid on the top of the agar coated dish, plunge the tube straight into the agar. Rotate the tube and take it out to release the plug from the agarose bed.
Verify the presence of the agar plug at the end of the tube. For long term imaging, cut three to five holes into the tube at each cardinal direction, at least five millimeters above the end of the fish by placing a razor blade perpendicular to the axis of the tube and make an incision into the tube at 30 degrees until reaching the mounting media. Then, make a second incision at 180 degrees to create a hole and roll the tube to visualize the cuts clearly.
Transfer the mounted embryo into a 1.5 milliliter microcentrifuge tube containing embedding media until ready to image. Mount the tube in the sample holder and fill the imaging chamber with embedding media. Place the sample holder on the stage with the sample dipping into the chamber.
Check the health of embedded fish by visually assessing the heart rate, and if the heart beat is too slow discard the sample. Always use the same sample position for reproducible imaging and rotate the fish so that both eyes are in the focal plane. Then, rotate the fish approximately 20 to 30 degrees counterclockwise for 48 hours post fertilization imaging.
When the fish has adapted to the laser power, select the illumination side that gives the best image quality. At each Z plane, record four to five heartbeats at 300 frames per second, or more. To record the beating heart move the sample stepwise through the light sheet and use a Z spacing of one to two micrometers to cover the entire depth of the heart.
Load the 4D file into a 3D rendering software to explore the data and generate movies of the rendered zebrafish heart. At 48 hours post fertilization the heart has just undergone looping and has two chambers, the ventricle and the atrium, but has yet to develop the valves. The different heart structures such as ventricle, atrioventricular canal, atrium, inflow tract, and outflow tract are easily distinguishable.
The data indicated precise beating and revealed complex interactions between the heart's two cell layers, the myocardium, a single cell muscle layer contracting and generating force, and the endocardium, a single cell layer connecting the heart to the vasculature. The most important is to always verify sample health and consistent heartbeat under a stereoscope. While conventional techniques often affect the heart shape or function, our methods deliver us dynamic unperturbed data of the beating heart that helps us understand the essentials of the early embryonic heart.
