Light Sheet Microscopy Sample Preparation: Mounting Live Zebrafish Embryos for Long-Term Imaging

- First, prepare agarose by adding E3 medium, which supplies calcium to the dechorionated embryos to molten agarose. Next, add MS222 to anesthetize embryos. Cool the agarose mix to 38 degrees Celsius, close to solidifying. Select a few dechorionated embryos expressing fluorescent proteins, and transfer them into the agarose mix.

Next, fully insert a plunger into a glass capillary and draw up one embryo, making sure that it enters head first. Once the agarose solidifies, use tape to hold the capillary upright in a beaker containing E3 medium. The open capillary bottom promotes gas exchange for the embryo sample.

Place the capillary in the sample holder of a light sheet fluorescence microscope. The laser forms a thin light sheet, allowing a detection objective-- positioned perpendicular to the sample-- to capture a cross-sectional image. In the example protocol, we will mount zebrafish embryos to image the developing eye.

Begin with preparing the agarose. Melt a prepared 1 milliliter aliquot of 1% low melting point agarose in E3 medium at 70 degrees Celsius. After about 15 minutes, transfer 600 microliters of molten agarose into a fresh, 1.5 milliliter tube, and add 250 microliters of E3 medium, 50 microliters of 0.4% MS222, and 25 microliters of vortexed fluorescent bead stock solution.

Place the tube in a heating block at about 39 degrees Celsius, to let the agarose approach its jelling point. Next, push Teflon-tipped plungers down to the bottom of 1 millimeter inner diameter glass capillaries. Prepare five of these.

Now, briefly vortex the agarose mixture and then, transfer five embryos with a minimal amount of liquid into it. Then, insert a capillary and aspirate one embryo, head first.

The head must enter before the tail and there cannot be any air bubbles. Draw up enough solution so there is about two centimeters of agarose above the embryo and 1 centimeter of agarose below it.

Draw one embryo into each capillary before proceeding. This step requires some practice to master. Prepare for it using non-valuable embryos.

When the agarose solidifies completely, store the samples in E3 medium by supporting the capillaries to a beaker wall with plasticine, with the bottom opening in solution. Now, assemble the sample holder. First, insert two plastic sleeves of the right size against each other into the stem.

The slits on the sleeves must face outwards. Next, attach a clamp screw and insert the capillary through the holder, until the black color band becomes visible on the other side. Avoid touching the plunger.

Then, tighten the clamp screw. Next, push a centimeter of agarose out from the capillary and trim it off. Then, insert the stem into the sample holder disk.

Before loading the sample disk, check that the stage is in the uppermost position in the control software. Then, use guiding rails to glide the holder and sample down into the microscope. Now, image the chosen sample as described in the text protocol.