This protocol enables a simultaneous acquisition of multiple embryos within one imaging session. While sample preparation is adapted for multi-point acquisition using a high content analyzer, samples prepared using this protocol can be imaged on any inverted microscope capable of multi-point acquisition. To begin, organize two rows of 15 to 20 embryos on a clean section of great plate using an egg picker tool.
Align the embryos on their ventral side in the same orientation with respect to anterior and posterior ends. Adhere a spacer onto a coverslip and streak 10 microliters of heptane glue down the exposed coverslip glass inset. While the glue dries, use a razor blade to cut a rectangle around the two rows of embryos.
Cut a second rectangle next to the first and remove the second rectangle with a straight edge side of a stainless steel spatula. Carefully slide the straight edge of the spatula under the first rectangle and remove it. Then, place the cutout with embryos on the external side of a 3.5 centimeter dish.
Under a stereo microscope, lower a prepared coverslip over the embryos and gently press the two rows of embryos onto the glue. If not performing microinjection, fill the silicone spacer halfway to the top with one-to-one halocarbon oil 700 to halocarbon oil 27. Line the bottom edges of a four-slide plate adapter with double-sided tape.
This will prevent the slide from moving during imaging. Ensure not to place the tape in the path of the objective lens. Mount the coverslip on the four-slide plate adapter.
If microinjecting the embryos, place the coverslip with embryos over a slide to prevent the bottom of the coverslip from becoming dirty during desiccation and microinjection. Desiccate the embryos for five to 10 minutes, then immediately cover them with one-to-one halocarbon oil 700 to halocarbon oil 27, filling the silicone spacer halfway to the top. Load two or three microinjection needles with approximately two microliters of injectant using an extended loading tip.
Mount a needle onto the microinjector and depress the plunger halfway to the tip, increasing the air pressure inside of the glass capillary. Lower the glass needle onto a glass slide and cut the tip of the needle with a new number nine razor, cutting at a 45 degree angle to produce a sharp open tip. The injectant should flow down to the bottom of the tip without flowing out of the needle.
Carefully add 20 microliters of halocarbon oil over the tip of the needle and re-cut it opened at a 45 degree angle. The injectant should begin to flow rapidly, so remember to adjust pressure by retracting the plunger. Use the micromanipulator to position the needle at the center of the field of view.
Then lift the needle and remove the glass slide from under it. Place the embryos on the stereo microscope stage, under the microinjection needle and focus on them at 50 times magnification. Lower the needle until it comes into the same focus plane as the embryos.
Moving the slide with one hand and operating the microinjector with the other, inject one row of embryos. Raise the needle and rotate the slide 180 degrees to expose the second row of embryos to the microinjection needle. Lower the needle and inject the second row of embryos.
This protocol was used to examine the role of microtubules in endoplasmic reticulum reorganization during mitosis in the Drosophila embryo. The effects of several microinjected drug treatments on ER reorganization were quantitatively compared. Colchicine, which prevents new microtubule polymerization, was found to drastically reduce the localization of the ER to spindle poles during mitosis.
Time lapse imaging data was acquired for 32 embryos. Mean and max intensity measurements were analyzed from 12, 800 regions of interest using a custom MATLAB script. When attempting this protocol for the first time, remember not to take too long on any one step, otherwise you risk missing your developmental stage that you want to image.
Practice this protocol step-by-step before attempting any experiments. Our method of sample preparation facilitates a simultaneous acquisition of multiple embryos during one experiment, thus generating enough experimental replicates for quantitative analysis. This protocol can help developmental embryology transition from qualitative to quantitative imaging experiments.
The ability to image multiple embryos within one imaging session also eliminates experimental noise between replicates.
