Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

1. Preparing molds and mounting agarose

1. Create a mold with the dimensions shown in Figure 1 using either a 3D printer or traditional mechanical workshop tools.
2. Pipette about 1.5 mL of melted 1 % agarose into a 35-mm plastic mounting dish. Place the plastic mold in the liquid agarose, taking care to avoid trapping air between the mold and the agarose. Place the dish at 4 °C, wait till the agarose hardens (this takes about 5 min), then remove the plastic mold.
3. To store the mounting dish for later use, cover the dish with its lid, then wrap both the dish and the lid tightly with parafilm. The mounting dish can then be stored lid side facing down for up to 5 days at 4 °C.
4. Prepare in advance 1 mL aliquots of 0.7 % low melting point agarose in 1.5 mL Eppendorf tubes to use on the day of mounting.
   NOTE: The agarose wells of the mounting dish will deform over time as water evaporates if parafilm is not wrapped around the dishes tightly.

2. Obtaining Embryos for the Photoconversion

1. Incross the Tg(fli1a:gal4FF^ubs, UAS:kaede) line and grow approximately 200 embryos in embryo medium in the dark at 28.5 °C. For more details on how to cross zebrafish lines, please refer to the JoVE Science Education Database¹¹.
2. After 5 h but before 24 h, treat embryos with 1-phenyl-2-thiourea (PTU) to prevent pigment formation (0.003 % PTU in embryo medium).
3. About 1 h before the start of photoconversion, screen embryos under a fluorescent stereoscope and select 5 healthy embryos that appear to express the brightest green fluorescence. The use of low-intensity light when visualizing embryos is recommended to avoid exposure of the embryos to ambient light in order to avoid photoconverting Kaede in non-specific cells.

3. Embedding

1. Prepare 10 mL of mounting media: 0.02 % tricaine and 50 mM BDM in embryo medium.
2. Add 2 mL of mounting medium to the mounting dish and allow 15-30 min for the solution to diffuse into the agarose.
3. In the meantime, melt a 1 mL aliquot of 0.7 % low melting point (LMP) agarose by placing the tube in a 70 °C heat block for about 5 min.
   1. Wait for the LMP agarose to cool slightly, then add 25 µL of 8 mg/mL tricaine solution and 40 µL of 1M BDM solution to the tube of low melting point agarose. Mix by pipetting up and down, then transfer the tube to a 38 °C heat block to keep the LMP agarose in its liquid state.
4. In a Petri dish with embryo medium, carefully dechorionate the pre-selected embryos under the stereomicroscope using forceps, taking care not to damage the embryos.
5. Transfer the embryos to a separate dish containing 2 mL of mounting media.
   1. When the hearts of the embryos are stopped (takes about 5-10 min), transfer the embryos to the mounting dish and arrange embryos in the wells using forceps so that they lie at a 25 degree angle, tails pointing towards the deeper part of the well, yolk facing up.
   2. When embryos are roughly in place, remove the media, embed the embryos in ~200 µL of 0.7 % LMP agarose containing tricaine and BDM, and readjust the embryos' positions, tilting the embryos to the left or right as necessary.
6. Wait until the LMP agarose sets (takes about 5 min), then add mounting medium to the dish. The embryos are now ready to be photoconverted.
   NOTE: The tricaine and the BDM are used to anesthetize the embryos and to stop the embryos' heart, respectively. Mounting media can be prepared the day before imaging, but make sure to protect the media from light.
   NOTE: The correct positioning of the embryos is important to allow a clear laser path to the cells one wishes to photoconvert. Position the head of the embryo close to the start of the well so that laser light does not have to travel far to reach the embryo, and so that the embryos can be easily unmounted after photoconversion.

4. Photoconversion

1. On an upright confocal microscope (like Leica SP8) equipped with a 405 nm, 488 nm and 561 nm laser source, locate the embryo's heart using transmitted white light (brightfield) and an objective lens (like Leica HCX IRAPO L, 25 ×, N.A. 0.95 objective). Check for red Kaede fluorescence using the 561 nm laser (i.e., DPSS 561 laser, at ~5 % intensity, detector set at 589-728 nm). Then, visualize the endocardium by using the 488 nm laser (i.e., 30 mW multiline Argon laser at ~5 % intensity, detector set at 502-556 nm).
2. Enter "FRAP" mode on the microscope acquisition software.Select ~1 % laser power for both 488 nm and 561 nm lasers.
3. Focus on the plane of interest, select a region of interest (ROI), then photoconvert cells using the 405 nm diode laser at 25 % laser power, scanning over the ROI three times. Should insufficient Kaede be photoconverted in the ROI, scan the 405 nm laser over the area three times again. Repeat this step for all ROIs.
4. Return to the "TCS SP8" mode on the software and acquire a z-stack, using the 561 nm and 488 nm lasers sequentially. Make sure to select the 'bidirectional' option to increase imaging speed.
   NOTE: How long it takes to photoconvert and image each embryo varies. Heart beat is known to be important for normal heart valve development, so avoid keeping the heart stopped for more than 30 min, even if it means you do not have time to photoconvert all 5 embryos.
   NOTE: During the photoconversion process, use PMT detectors, while after photoconversion, it is recommended to use hybrid detectors set to 'counting' mode. This is because the hybrid detectors are more sensitive and are capable of producing better quality images, but are easily damaged by overexposure.

5. Unmounting photoconverted embryos

1. After photoconversion, use a glass pipette to press down slightly just above the head of the embryo to break the LMP agarose, then gently suck up the embryo.
   1. Eject the embryo from the glass pipette into a 35-mm Petri dish containing embryo medium with PTU (to wash away BDM containing medium).
   2. Transfer the embryo to a well in a 6-well plate containing embryo medium with PTU. During this step, make sure to keep note of which embryo goes into which well, as this is essential to correlate the position of photoconverted cells at later developmental stages.
2. Now that the embryos are removed from the BDM containing medium, their hearts should start beating again in about 5 min. Return embryos to the dark at 28.5 °C to allow embryos to continue to develop normally.

6. Imaging photoconverted cells at later embryonic stages

1. At the desired stage, stop the heart and re-embed the embryos like under Step 3 of this protocol, with the important difference that embryos must be treated with BDM in separate, marked dishes to keep track of embryos.
2. For embryos up to 62 hpf, acquire z-stacks of the endocardium using 561 nm and 488 nm for red and green fluorescent signals, respectively. For embryos at stages older than 62 hpf, use a multiphoton laser set to 940 nm to image green Kaede instead of the 488 nm laser.

7. Image analysis for embryos under 48 hpf

Note: Tissue movements of the AVC can be difficult to analyze due to its complex three-dimensional structure. To quantify the length of a photoconverted region in the AVC between two non-photoconverted regions or the length of a non-photoconverted region in the AVC between two photoconverted regions, it can be desirable to map the three-dimensional structure onto a two-dimensional map. Images of photoconverted embryos obtained prior to 48 hpf can be analyzed using the following method.

1. Import the acquired images in Matlab or any similar software.
2. Delimitate manually the ROI, i.e. the endocardium, and apply a mask to remove the signal outside this region.
3. Apply an intensity threshold on the image to identify the points on the endocardium and plot them in three-dimensions. Isolate the points corresponding to the narrowest and most straight part of the AVC, by erasing manually the points of no interest on the atrium and the ventricle using the erase command in the software.
4. Use a cylinder to fit the points representing the AVC so obtained.
5. Define a reference system to compare several samples. For example, adopt the center of mass of the AVC points as the origin of the reference system and the axis of the fitted cylinder as the z-axis.
   1. Make sure that positive z-positions correspond always to the same side of the heart (ventricular or atrial).
   2. Project the AVC points on the x-y plane and use an ellipse to fit them.
   3. Rotate the endocardial points around the z-axis so that the major axis of the ellipse and the y-axis of the reference system are parallel and positive y-values correspond to the internal side of the AVC with respect to the embryo.
6. To segment the AVC, cut the three-dimensional dataset with some planes obtained by rotating the half-plane {y=0, x>0} in the z-direction.
   1. Project the intensity of the neighboring pixels on these planes and define the endothelium in a consistent manner. For example, draw manually a line from the atrial to the ventricular side of the AVC at half thickness of the endothelium.
   2. Interpolate the points of the lines representing the endothelium with a three-dimensional spline using the spline toolbox to represent the AVC with a surface.
      NOTE: The number of the cutting planes determines both the accuracy of the segmentation and the time consumption of this step. Usually, 8 planes are enough to achieve good results.
7. Define the azimuthal position of each point on the AVC surface using cylindrical coordinates.
   1. At each azimuthal position define a parametric curve along the surface of the AVC by calculating the arc length on the surface between consecutive points.
   2. For each parametric curve, define the point on the curve closest to the center of the AVC to zero. The points of the AVC are thus fully described by their azimuthal position and their position on the parametric curve.
8. Unfold the AVC into a two-dimensional image by using the parametric position of the points.
9. Find the edges of the region to quantify as the edges of the binary image thresholding the ratio between the intensities of the photoconverted and the non-photoconverted channels. Manually correct the result if necessary.
10. Calculate the arc length between the edges to compute the length of the tissue as a function of the azimuthal position.