In the early Drosophila embryo, many organelles undergo large scale motion. Our protocol describes how to monitor this motion using fluorescent dyes developed for cell culture. Compared to fluorescent proteins, commercially available fluorescent probes are brighter and span a wider range of the spectrum.
They allow for multiplex colabeling and can be used in any genetic background. It takes practice and trial and error to know how long to desiccate embryos, so that they either burst during injection or dry up and stop developing. To begin, start preparing the injection needles by placing the capillary in a capillary holder on the needle puller and securing it using wing nuts.
Align the center of the capillary with the heating element. According to the manufacturer's instructions, choose appropriate settings on the needle puller. Using a dissecting microscope, perform the quality control test.
Press the needles into the putty horizontally with the needle tips suspended in the air. Cover the needles with the lid and store until use. Using needle loading tips attached to a micropipette, draw one microliter of the injection solution into the tip without trapping air bubbles.
Using gloves, hold the needle in one hand with its tip pointing away and insert the loading tip into it. Dispense the liquid near the needle tip, remove the pipette and hold the needle vertically until the liquid flows to the tip. Cover the container with aluminum foil without damaging the needle tips to prevent the bleaching of the dyes.
Using a transfer pipette, or a micropipetter, place a small drop of heptane glue at the center of a rectangular cover slip, and allow it to evaporate. For removing the chorion mechanically, cover the appropriately aged embryo plate with a thin layer of halocarbon oil 27 to turn the eggshell transparent. View the plate under a dissection microscope with trans illumination to confirm the embryo stage and select the embryos in cleavage stages.
Using fine tweezers, pick up an embryo of the desired stage by grabbing the dorsal appendages and transfer it to the prepared glass slide covered with double sided tape. Place the embryo on the tape and minimize the transfer of oil. Roll the embryo gently across the surface of the tape by nudging with the side of the tip of the tweezers without poking it directly.
Continue rolling the embryo until the chorion adheres to the tape and cracks. After the chorion cracks, keep rolling the embryo to separate it from the chorion, which remains stuck to the tape. Roll the embryo back onto the chorion, which is less adhesive than the tape.
Gently rub the embryo with the tweezers until it attaches to the tweezers for transfer. Then, transfer the embryo to the cover slip and bring it in contact with the glue to hold the embryo in place, separating it from the tweezers. Adjust the orientation of the embryo on the cover slip.
Let the embryos desiccate for five to 12 minutes by placing the cover slip with embryos into the desiccation chamber and sealing it. Take off the cover slip from the chamber and put a drop of halocarbon oil 700, covering the embryos to prevent further desiccation. Examine them under the dissecting scope and proceed to microinjection if the embryos were desiccated properly.
Place the 4X objective into the light path and using the focus knob on the microscope, adjust the embryos into focus. Move the embryos horizontally into the center of the field of view using the stage controls. Keep the embryos at the edge of the field of view and pan away in preparation for lowering the needle.
Staying with the same focal plane, lower the needle tip and ensure it is visible in the field of view along with the embryos. Check that needle is functioning by dispensing some of the injection solution into halocarbon oil 700. If successful, a bubble of solution will appear in the oil near the needle tip.
Using the stage controls, move the embryo towards the needle tip until the tip gently punctures the embryo and enters into it. Simultaneously, begin the flow of injection solution until a transient clear spot appears at the site of the needle tip, indicating successful solution transfer into the embryo. Secure the cover slip on the confocal microscope using the slide holder.
Use caution as the cover slip is fragile. Inspect the embryos using the epifluorescence function on the confocal microscope with a 40X objective and ensure that the fluoro 4 in the embryo is visible before proceeding. For live imaging, during the syncytial stages, set the image size of 512 by 512 pixels, line average of three, and a frame rate of 0.1 frames per second.
After the BODIPY 493 503 dye was injected, it was localized at the site where the needle tip was inserted and then diffused adjacent to the injection site. After 24 minutes, the dye has diffused past the midpoint of the embryo. The organelle motility was monitored by injecting the embryo with BODIPY 493 503 and LysoTracker Red.
The particle image velocimetry analysis revealed that the embryonic contents converged on the central region of the embryo, where the cytoplasm is flowing into the embryo's interior. Labeling of the ER, using ER-Tracker Green, provides a vivid resolution of the nuclear envelope, allowing visualization of major cell cycle stages. The embryo was injected with MitoView 633 at the blastoderm stage and imaged four hours later.
The image shows a neuroectodermal cell of an embryo in germ band extension. A cellularizing embryo was injected with a mixture of BODIPY 493 503 and SiR Tubulin, which shows that the lipid droplets move bidirectionally along microtubules. The embryo must be partially desiccated so that liquid can be injected.
Too little desiccation will cause the embryo to leak when injected, too much desiccation will kill the embryo. Videos of moving organelles can be characterized via diverse image analysis techniques. Particle tracking reveals the behavior of individual organelles, particle imaging velocimetry reveals the bulk flow.
