Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

This method can help answer key questions in the developmental cardiac mechanobiology field, such as the modulation of shear stress on cardiac trabeculation via notch signaling using a 4D light sheet microscope. The main advantage of this technique is that it illuminates a thin section of sample with a five micron plane, leading to less photo bleaching and photo damage. In addition, our computational algorithm allows us to reconstruct a 4D beating zebrafish heart.

The implications of this technique extend toward the diagnosis of congenital cardiac diseases as it allows for the invivo sequential visualization of the cardiac trabeculation in an early developmental stage on through to the later phenotype. Generally, people who are new to this technique will find that thinking in three dimensions over time is difficult to imagine. In addition, it takes time to create the system and accurately align it.

To begin, separate the male and female zebrafish in the the breeding tanks by using a divider. Then, lift the divider and allow the male and female zebrafish to breed. Collect the zebrafish embryos and inject morpholino oligo into the embryo to inhibit trabeculation.

Next, prepare a one percent agarose gel by adding one gram of agarose to 100 milliliters of distilled water and heat it until all of the agarose is dissolved. Allow the agarose to cool and then use a 20 microliter pipet to load a small plastic tube with one of the prepared embryos and agarose. Secure the tube onto the microscope stage and use the ten x objective.

Adjust the objective using the knob so that the top of the embryo is in focus. Take images of the entire fish embryo over multiple cardiac beating cycles using a light sheet thickness of five microns. Collect 500 xy frames with an exposure time of ten milliseconds.

Then, move the stage one micron in the z access to a new layer and repeat the imaging procedure. Continue imagine 500 frames per z plane until the entire heart is fully imaged. Next, open the image processing software and begin to stack the images into 3D.

To accomplish this, first click on open data. Here, select all of the images that are to be stacked into 3D and then select load. Then add in the voxel size of 0.65 by 0.65 by one micron and click on okay.

Inputting the correct voxel size is critical for accurate analysis of the 3D images. Now, click on the multiplaner viewbox and visualize the 3D image. Right-click the blue slice one dot tiff box, select display, and then click on volran.

In the edit menu, select options, and then edit color map. Adjust the color using the boxes and then click on okay. While still in the image processing software, click on file, and open the time series data.

Select the 3D tiffs that were just made by clicking on load, and enter the same voxel size as before. Right-click the blue slice one dot tiff box, and then select display followed by volran. Now, click on edit.

Select options. And then select edit color map. Adjust the color using the boxes and then click on okay.

Next, right-click on the time series control, click movie maker, and press the play button in order to watch the movie. To finish up, add a file name, frame size, frame rate, quality equal to one, and enter monoscopic. Then click apply.

Finally, export the video. Biomechanical forces, such as hemodynamic shear stress, are intimately involved in cardiac morphogenesis. Here, light sheet fluorescence microscopy was used to visualize the trabecular ridges protruding into the ventricular lumen at 75 hours post fertilization.

At 100 hours post fertilization a trabecular network can clearly be seen. In an embryo treated with the gata1aMO microinjection, hematopoiesis and viscosity are reduced by 90 percent. This decreased viscosity attenuated hemodynamic shear stress, resulting in a delayed initiation and a less dense trabecular network.

This delay and density of the trabecular network was able to be recovered by up regulating notch related gene expression, rescuing trabecular formation at 75 hours post fertilization and at 100 hours post fertilization. Thus, gata1a morpholino oligonucleotides reduced hemodynamic forces leading to down regulation of notch signaling, whereas Nrg1-mRNA rescue up regulated notch related genes and reinitiated trabeculation. Once mastered, this imaging technique can be completed in approximately two hours if performed properly.

After its development, this technique paved the way for researchers in the field of cardiac development to study the factors in the notch gene, which are responsible for the proper formation of trabeculation in zebrafish. After watching this video, you should have a good understanding of how to acquire images using selective plane illumination microscopy and create 4D image files. Don't forget that working with lasers can be extremely hazardous.

And precautions, such as wearing appropriate eyewear, should always be taken during this procedure.