Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

The overall goal of this procedure is to mount a zebra fish embryo for long-term in vivo light sheet microscopy. This is accomplished by first cleaning a fluorinated, ethylene propylene or FEP tube and coating it with Methylcellulose. Next, a zebra fish embryo of interest is coated.

Then the embryo is transferred first to melted agarose, and then to the coated FEP tube. Finally, the bottom of the tube is closed with an aros plug and the orientation of the mounted embryo is carefully checked. Ultimately, results can be obtained that show the unperturbed development of zebra fish embryos over several days with high spatial and temporal resolution through in vivo light sheet microscopy.

Well, the main advantage over existing techniques is that we can now observe super fish embryos over several days and not just a few hours. This method can provide valuable insights into the long-term embryogenesis of the zebra fish using light sheet microscopy. It can also be applied to other small organisms like small marine organisms.

It can also be used with other microscopy techniques such as optical projection tomography, widefield microscopy, or confocal microscopy. Individuals new to this method might struggle because the protocol involves some time critical steps and judging the final position of the embryo requires some expertise To prepare an FEP tube First, clean it by flushing it repeatedly with one molar sodium hydroxide. Then transfer the tube to a 50 milliliter centrifuge tube filled with 0.5 molar sodium hydroxide and ultrasonic eight for 10 minutes.

Transfer the polymer tube to a small basin with distilled deionized water and flush it. Then follow with a 70%ethanol flush before transferring it to a fresh centrifuge tube. With ethanol ultras.

Sonicate the tube for 10 minutes before transferring it to a small basin with distilled de ionized water and flushing it.Again. Cut the clean FEB tube into three centimeter long pieces and straighten them if necessary. Store the cleaned tubes in a fresh 50 milliliter tube filled with distilled deionized water.

Prepare Trica stock solution. 3%Methylcellulose and both 1.5%and 0.1%Aros according to the text protocol. Melt 1.5%agarro and pour it into a Petri dish to form a layer with a thickness of about two millimeters.

Collect zebrafish embryos at the desired stage that have positively expressed the fluorescence gene of choice and transfer them to a fresh dish of E three under a stereoscope. Using two sharp forceps, carefully coate the embryos by using one forceps to hold corion and the second forceps to tear it open and pull it off to carry out multi-layer mounting. With gloved hands, attach a blunt end cannula to a syringe.

Carefully insert the cannula about three millimeters into one end of a cleaned piece of FEP tube. Next, dip the free end of the FEP tube into 3%Methylcellulose and fill the tube with the solution. Then slowly push on the syringe to release the methylcellulose using E three medium, repeat the dipping and expelling steps.

Meanwhile, in a heat block, heat up one aliquot of 0.1%aros to 70 degrees Celsius. Vortex the reaction tube briefly before transferring it to a heat block set to 38 degrees Celsius. After heating, allow the tube to cool down briefly and add trica to a final concentration of 133 to 200 milligrams per liter and vortex.

Again, using a glass paster pipette. Transfer a coated embryo to the reaction tube with the preheated aros transferring as little E three as possible. With the FEP tube and attached syringe gently take up a small amount of aros before taking up the embryo and fill the tube completely with aros.

The embryo should be positioned at either end of the tube with the head pointing towards the opening. To avoid leaking the contents, keep the tube in a horizontal position to plug the tube. Use a razor blade to cut it off the cannula.

Then holding the tube perpendicular slowly stick the end of it through the entire layer of 1.5%aros and rotate it slightly. To avoid drying, keep the mounted specimen in a 1.5 milliliter reaction tube filled with E three medium Before imaging. Verify that the fish inside the FEP tube is positioned straight, and then its head sits directly on top of the plug for imaging.

Add trica to the medium inside the imaging chamber. Shown here is a one day post fertilization transgenic. K-G-R-L-G-F-P zebrafish embryo imaged over the course of two days to observe the development of the vasculature.

Zebrafish is not restricted by the mounting medium and can develop normally. For example, its head raises. The tail extends and vascular sprouting appears unhindered.

This technique can be done in 10 minutes if performed properly and all material is readily available. Remember to perform all the major steps without any delay. Keep all the solutions clean since dirt can impair the final image quality.