The overall goal of this procedure is to visualize calcium during egg activation in Drosophila melanogaster. This method can help answer key questions in developmental biology, such as what the source, dynamics, and downstream roles of calcium are at egg activation. The main advantages of this technique are to allow higher spatial resolution, physical manipulation of the mature egg before activation, and testing the role of egg activation without fertilization.
Generally, individuals new to this method will struggle with isolating the mature eggs without damage, and applying activation buffer to the mature egg without losing the sample. To begin this procedure, set the magnification of the dissecting microscope to 4X. On the first coverslip, separate the two ovaries by tearing the oviduct with a pair of closed forceps and a dissecting probe, taking care not to puncture any egg chambers.
Next, insert a standard dissecting probe into the center of the ovary, next to the closed forceps. Gently drag the dissecting probe through the ovary, towards the posterior pole, where the mature eggs are located. Once the mature eggs are visible on the coverslip outside of the ovary, maneuver three to five of them in a line in the center.
Next, remove the rest of the ovarian tissue by dragging it away from the aligned eggs. Then, remove excess oil by dabbing it with the corner of a medical wipe. Draw a crosshair on the coverslip with a marker to locate the sample under the microscope.
After that, leave the prepared egg chambers on the coverslip to rest for five to 10 minutes to allow the eggs to settle in the oil. Bring to the imaging facility the prepared coverslips, room-temperature activation buffer, and glass pipettes. Select an objective suitable for imaging the entire mature egg.
Then, mount the first prepared coverslip onto the microscope stage. Next, select a field of view of a mature egg chamber for activation. Set the imaging parameters for standard GFP excitation and emission.
After that, set up a bright field view in order to visualize and orientate the mature egg and displaced oil. Now, select the lowest Z-plane where the mature eggs are visible. And set a full Z-stack to be taken for 40 micrometers at two micrometers per step.
Then, set the time series to capture the images for 30 minutes. In this procedure, start image capture, and acquire one to two full Z-stacks to show the mature egg prior to activation. Next, withdraw approximately 300 microliters of activation buffer into a glass pipette.
Position the tip of the glass pipette containing activation buffer at a 45-degree angle above the coverslip, and slowly move the pipette into the beam path, until it is directly above the mature egg being imaged. Add one to two drops of activation buffer in less than five seconds, to displace the oil. The addition of one to two drops of activation buffer needs to be done at the correct angle, gently, and quickly.
Whilst adding activation buffer during image acquisition, check the bright-field channel to ensure that the oil has been displaced. Once the oil is successfully displaced, allow the time series to run until completion. To build a projection of the acquired data, select a start Z-slice and a stop Z-slice for the whole section.
Afterward, select a projection type, for example, maximum intensity. Next, check the tick box All time frames to apply the selected settings to the whole series. To adjust the image brightness and contrast, select Image, then Adjust, followed by Brightness/Contrast.
To export the time series as a movie, select File, then Save As, followed by AVI. Then, select the frame rate. Shown here is the time-series of an ex vivo mature Drosophila egg expressing a genetically encoded calcium indicator following the addition of activation buffer.
The rise in cytoplasmic calcium originates at the posterior pole, and then propagates with an average velocity of approximately 1.5 micrometers per second. Initiation of the wave is typically observed within three minutes of the addition of activation buffer. Following activation, the intracellular calcium levels of the mature egg recover.
In Drosophila, co-visualization of the calcium wave and actin cytoskeleton can be achieved using this ex vivo activation assay. Actin appears to be changing over time, as indicated by the white arrowheads. The lack of calcium signal detection in the center of the mature egg is due to movement of the sample during image capture.
Once mastered, this entire technique can be done less than an hour, if performed properly. While attempting this procedure, it's important to remember to take your time when maneuvering the egg chambers, and when setting up the parameters for acquisition. This procedure can be adapted to visualizing other flourescently labeled proteins, organelles, or calcium indicators at egg activation.
This can help to address questions regarding the source of calcium and the downstream effects.
