Light Sheet Microscopie Monstervoorbereiding: Montage van levende zebravisembryo's voor langdurige beeldvorming

- 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 o