We are grateful to Laura Bampton, Alex Davidson, Richard Parton, Arita Acharya for assistance during the preparation of this manuscript; Mariana Wolfner for discussions on egg activation; Matt Wayland for imaging support; and Nan Hu for general support in the laboratory.
This work was supported by the University of Cambridge, ISSF to T.T.W. [grant number 097814].

Note: Carry out all steps at room temperature unless stated otherwise.
1. Preparing for Dissection
Note: The steps described here are in accordance with E. Gavis, Princeton University &#38; Weil et. al. 18. Perform the following steps two days before imaging.
<ol>
	<li>Take a fly vial containing approximately 10 ml of solid food and add a small amount of dried yeast to one corner, less than 1 g is sufficient. 
	Note: For information about fly vials or food see &#39;Drosophila Maintenance&#39; 19.</li>
	<li>Add 1 - 3 drops of water to hydrate the yeast.</li>
	<li>Set the vial aside at a 45&#176; angle and allow 3 - 5 min for the yeast to take up the water.</li>
	<li>While the yeast is absorbing the water, select a bottle of flies expressing UASt-myrGCaMP5 20 driven by tub-VP16GAL4 (referred to as GCaMP5) that have recently emerged (a myristoylated form of the GCaMP5 was chosen to provide a high signal to noise ratio through tethering the GECI to the membrane in the egg).</li>
	<li>Anaesthetize the flies in the bottle with CO2 gas. Be sure to keep the bottle inverted so as not to lose flies in the wet food.</li>
	<li>Tip the anesthetized flies onto a CO2 pad and sort 10 - 15 females and 5 males using a paintbrush under a dissecting microscope. Note: It is standard to include males to provide healthy females for dissection.</li>
	<li>When the vial from step 1.3 is ready, add the selected flies to the vial. Take care to keep the vial horizontal until the flies become active.</li>
	<li>Place the vial at 25 &#176;C for approximately 36 hr.</li>
	<li>Return the remaining flies from the CO2 pad to the bottle or discard.</li>
</ol>
2. Dissecting Drosophila Ovaries
Note: The steps described here are in accordance with Weil et. al. 18.
<ol>
	<li>After 36 hr, anaesthetize the flies from the yeasted vial with CO2.</li>
	<li>Once the flies inside the vial are anesthetized tip them onto the CO2 pad to be sorted with a paint brush under a dissecting microscope.</li>
	<li>Select a female.</li>
	<li>Isolate the two ovaries from the female. For full detailed protocol see Weil et. al. 18. In summary:
	<ol>
		<li>Place a small drop of oxygenated halocarbon oil (series 95) on a No. 1.5, 22 x 40 mm coverslip.</li>
		<li>Grasp the female with forceps at the anterior of the abdomen.</li>
		<li>Holding the female under the dissecting microscope, use the second set of forceps to gently remove the posterior of the female.</li>
		<li>Wipe off the posterior tissue from the second forceps.</li>
		<li>Squeeze on the abdomen cavity with the second forceps from the anterior to the posterior of the abdomen. The two opaque ovaries should stick to the forceps when outside of the female.</li>
		<li>Touch the ovaries into the oil.</li>
		<li>Remove any tissue from the forceps by wiping off the forceps.</li>
		<li>Repeat these steps (2.4.1 - 2.4.7) to obtain three coverslips, each containing ovaries from one fly.</li>
	</ol>
	</li>
</ol>
3. Isolating Individual Mature Eggs
<ol>
	<li>Under a standard dissecting microscope with the magnification set to 4X, on the first coverslip from step 2.4, separate the two ovaries by tearing the oviduct.</li>
	<li>Introduce a pair of closed forceps into the center of a single ovary, taking care not to puncture any egg chambers.</li>
	<li>Insert a standard dissecting probe into the center of the ovary next to the closed forceps.</li>
	<li>Gently drag the dissecting probe through the ovary towards the posterior pole, where the mature eggs (stage 14 egg chambers) are located.</li>
	<li>Repeat steps 3.1 - 3.4 2 - 5 times depending on the size of the ovary and number of eggs. 
	Note: Addition of CO2 typically causes the deposition of a single egg which will appear larger with raised dorsal appendages than pre-deposited eggs. This egg should be discarded for imaging since it has already been activated inside the female. 
	Note: Mature eggs are likely to separate easily from ovarian connective tissue.</li>
	<li>If no mature eggs are visible outside of the ovary, continue gently teasing with the dissecting probe.</li>
	<li>Once the mature eggs are visible on the coverslip outside of the ovary, maneuver 3 - 5 in a line in the center of the coverslip. 
	Note: For best results, run the probe gently along the side of the eggs. 
	Note: Avoid pulling on the dorsal appendages as this can damage the egg. 
	Note: Depending on the future imaging set-up, align the eggs approximately 200 &#181;m apart perpendicular to the coverslip for maximum imaging area.</li>
	<li>Once aligned, remove the rest of the ovarian tissue by dragging it away from the aligned eggs using the forceps.</li>
	<li>Remove excess oil by dabbing with the corner of a medical wipe. Be careful not to touch the mature eggs with the medical wipe as mature eggs that are contacted will be lost.</li>
	<li>Draw a crosshair on the coverslip with a marker to simplify locating the sample under the microscope.</li>
	<li>Set the coverslip in a safe place and leave the prepared egg chambers on the coverslip to rest for 5 - 10 min. This allows for the egg to settle in the oil. Samples can be used up to 1 hr post dissection.</li>
	<li>Repeat steps 3.1 - 3.11 for the rest of the coverslips with ovaries on them.</li>
</ol>
4. Preparing for Imaging
<ol>
	<li>Bring prepared activation buffer 15 to room temperature. 
	Note: Activation buffer is a hypotonic buffer: 3.3 mM NaH2PO4, 16.6 mM KH2PO4, 10 mM NaCl, 50 mM KCl, 5% polyethylene glycol 8,000, 2 mM CaCl2, brought to pH 6.4 with a 1:5 ratio of NaOH:KOH. For more information see Mahowald, 1983 15. Aliquots of activation buffer can be stored at -20 &#176;C for months and at 4 &#176;C for up to 2 weeks.</li>
	<li>Carefully transport prepared coverslips, activation buffer and glass pipettes to the imaging facility. 
	Note: A wide-field, confocal, spinning disc or light sheet microscope can be used. We recommend using an inverted microscope. Our representative results were obtained using a confocal microscope.</li>
	<li>An objective suitable for imaging the entire mature egg should be chosen (here: 20X 0.7 NA).</li>
	<li>Mount the first prepared coverslip on to the microscope stage. Take care not to break the coverslips on the stage.</li>
	<li>Qualitatively select a field of view with a mature egg chamber for activation. Do not image punctured egg chambers or those with blebbing in the membrane. 
	Note: For steps 4.6 - 4.7, software commands will vary depending on microscope and manufacturer.</li>
	<li>Set imaging parameters for standard GFP excitation (488 nm) and emission (500 - 580 nm). Also set up a bright-field view in order to visualize and orientate the mature egg and displaced oil.</li>
	<li>Select the lowest Z-plane where mature eggs are visible and set a full Z-stack to be taken for 40 &#181;m at approximately 2 &#181;m per step. Set the parameters so there is no delay between Z-stack acquisition. Set the time series to capture for approximately 30 min.</li>
</ol>
5. Imaging Ex Vivo Egg Activation
<ol>
	<li>Start image capture.</li>
	<li>Wait for 1 - 2 full Z-stacks to be acquired. This shows the mature egg prior to activation.</li>
	<li>Withdraw approximately 300 &#181;l of activation buffer into a glass pipette.</li>
	<li>Positioning the tip of the glass pipette containing activation buffer at a 45&#176; angle above the coverslip, slowly move the pipette into the beam path until it is directly above the mature egg being imaged. The glass pipette should be 1 - 2 cm above the oil.</li>
	<li>Add 1 - 2 drops of activation buffer in less than 5 sec from the glass pipette. This should displace the oil. Avoid adding excess activation buffer or touching the egg with the glass pipette as it can cause displacement of the mature egg. Take care not to touch the coverslip with the pipette as this can cause a change in the focal plane or air bubbles to form in the immersion oil between the objective and coverslip.</li>
	<li>Whilst adding activation buffer during image acquisition, check the bright-field channel to ensure that the oil has been displaced by the activation buffer. 
	Note: This will first appear as a shadow due to the pipette breaking the beam path but will also result in the appearance of small oil droplets. Some oil droplets can remain associated with the mature egg which will affect experimental outcomes and image quality.</li>
	<li>If the oil is not displaced from the initial addition of activation buffer repeat steps 5.4 - 5.6.</li>
	<li>Once the oil is successfully displaced, allow the time series to run until completion. 
	Note: Due to swelling of oocytes at activation, some egg chambers will move dramatically out of the focal plane or field of view upon addition of activation buffer. In this situation, stop the acquisition and mount the next coverslip.</li>
	<li>After the image sequence has completed acquisition, save data.</li>
</ol>
6. Post-acquisition Image Processing
<ol>
	<li>Open the data set using Fiji (http://fiji.sc/Downloads#Fiji), or equivalent image processing software, and select the relevant files for analysis.</li>
	<li>To build a projection of the acquired data select: Image &#62; Stacks &#62; Z-project.</li>
	<li>Select a start Z-slice and a stop Z-slice, typically the whole section. Select a projection type, typically set to &#39;Max Intensity&#39;. Check tick box &#39;All Time Frames&#39; to apply the selected settings to the whole series.</li>
	<li>If more than one fluorescent channel has been imaged, use the Channels tool to enable movement between colors, make a composite, or make the image grayscale. Select Image &#62; Colors &#62; Channels tool.</li>
	<li>To adjust the image brightness and contrast select Image &#62; Adjust &#62; Brightness/Contrast.</li>
	<li>To export a single image, move timescale slider to relevant frame and select File &#62; Save As &#62; Jpeg, or any preferred format.</li>
	<li>Choose a location and title for the exported file, then save. 
	Note The frame rate from the imaging software will be required to give a time stamp on the image.</li>
	<li>To export the time series as a movie select: File &#62; Save As &#62; AVI, then select frame rate (~ 7 frames per second).</li>
	<li>Choose a location and title for the exported file, then save.</li>
	<li>To save the adjusted file in Fiji format, select File &#62; Save.</li>
</ol>

<ol>
	<li>Stricker, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+biology+of+calcium+signaling+during+fertilization+and+egg+activation+in+animals.">Comparative biology of calcium signaling during fertilization and egg activation in animals.</a> Dev Biol. 211 (2), 157-176 (1999).</li><li>Horner, V. L., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitioning+from+egg+to+embryo:+triggers+and+mechanisms+of+egg+activation.">Transitioning from egg to embryo: triggers and mechanisms of egg activation.</a> Dev Dyn. 237 (3), 527-544 (2008).</li><li>Gilkey, J. C., Jaffe, L. F., Ridgway, E. B., Reynolds, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+free+calcium+wave+traverses+the+activating+egg+of+the+medaka,+Oryzias+latipes.">A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.</a> J Chem Biol. 76 (2), 448-466 (1978).</li><li>Fujiwara, T., Nakada, K., Shirakawa, H., Miyazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+inositol+trisphosphate-induced+calcium+release+mechanism+during+maturation+of+hamster+oocytes.">Development of inositol trisphosphate-induced calcium release mechanism during maturation of hamster oocytes.</a> Dev Biol. 156 (1), 69-79 (1993).</li><li>Hafner, M., Petzelt, C., Nobiling, R., Pawley, J. B., Kramp, D., Schatten, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wave+of+free+calcium+at+fertilization+in+the+sea+urchin+egg+visualized+with+fura-2.">Wave of free calcium at fertilization in the sea urchin egg visualized with fura-2.</a> Cell Motil Cytoskeleton. 9 (3), 271-277 (1988).</li><li>Fontanilla, R. A., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+sperm-induced+calcium+wave+in+Xenopus+eggs+using+confocal+microscopy.">Characterization of the sperm-induced calcium wave in Xenopus eggs using confocal microscopy.</a> Biophys J. 75 (4), 2079-2087 (1998).</li><li>Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+Ca2++indicators:+using+genetics+and+molecular+design+to+understand+complex+physiology.">Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology.</a> J Physiol. 578, 55-67 (2007).</li><li>Douglass, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> New Techniques in Systems Neuroscience. , (2015).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19 (2), 137-141 (2001).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J Neurosci. 32 (40), 13819-13840 (2012).</li><li>Chen, T. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity.">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li><li>Wang, J., Wong, A., Flores, J., Vosshall, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain.">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112 (2), 271-282 (2003).</li><li>Kaneuchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+waves+occur+as+Drosophila+oocytes+activate.">Calcium waves occur as Drosophila oocytes activate.</a> Proc Natl Acad Sci U S A. 112 (3), 791-796 (2015).</li><li>York-Andersen, A. H., Parton, R. M., Bi, C. J., Bromley, C. L., Davis, I., Weil, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+and+rapid+calcium+wave+at+egg+activation+in+Drosophila.">A single and rapid calcium wave at egg activation in Drosophila.</a> Biol Open. 4 (4), 553-560 (2015).</li><li>Mahowald, A. P., Goralski, T. J., Caulton, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+activation+of+Drosophila+eggs.">In vitro activation of Drosophila eggs.</a> Dev Biol. 98 (2), 437-445 (1983).</li><li>Page, A. W., Orr-Weaver, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+meiotic+divisions+in+Drosophila+oocytes.">Activation of the meiotic divisions in Drosophila oocytes.</a> Dev Biol. 183 (2), 195-207 (1997).</li><li>Horner, V. L., Wolfner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stimulation+by+osmotic+and+hydrostatic+pressure+activates+Drosophila+oocytes+in+vitro+in+a+calcium-dependent+manner.">Mechanical stimulation by osmotic and hydrostatic pressure activates Drosophila oocytes in vitro in a calcium-dependent manner.</a> Dev Biol. 316 (1), 100-109 (2008).</li><li>Weil, T. T., Parton, R. M., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+individual+Drosophila+egg+chambers+for+live+imaging.">Preparing individual Drosophila egg chambers for live imaging.</a> J Vis Exp. (60), (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Biology+1:+yeast,+Drosophila+and+C.+elegans.+Drosophila+Maintenance.">Essentials of Biology 1: yeast, Drosophila and C. elegans. Drosophila Maintenance.</a> JoVE Science Education Database. , (2016).</li><li>Melom, J. E., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+a+NCKX+Eliminates+Glial+Microdomain+Calcium+Oscillations+and+Enhances+Seizure+Susceptibility.">Mutation of a NCKX Eliminates Glial Microdomain Calcium Oscillations and Enhances Seizure Susceptibility.</a> J Neurosci. 33 (3), 1169-1178 (2013).</li><li>Munro, E., Nance, J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+flows+powered+by+asymmetrical+contraction+transport+PAR+proteins+to+establish+and+maintain+anterior-posterior+polarity+in+the+early+C.+elegans+embryo.">Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo.</a> Dev Cell. 7 (3), 413-424 (2004).</li><li>Maruyama, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGG-3+regulates+cell-surface+and+cortex+rearrangements+during+egg+activation+in+Caenorhabditis+elegans.">EGG-3 regulates cell-surface and cortex rearrangements during egg activation in Caenorhabditis elegans.</a> Curr Biol. 17 (18), 1555-1560 (2007).</li><li>Wong, G. K., Woods, M. A., Szymczak, R. K., Gavlik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+normal+actin+cytoskeletal+reorganization+affects+cell+cycle+time+in+fertilized+sea+urchin+eggs.">Disruption of normal actin cytoskeletal reorganization affects cell cycle time in fertilized sea urchin eggs.</a> Mol Biol Cell. 15, 23 (2004).</li><li>Horner, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+calcipressin+sarah+is+required+for+several+aspects+of+egg+activation.">The Drosophila calcipressin sarah is required for several aspects of egg activation.</a> Curr Biol. 16 (14), 1441-1446 (2006).</li></ol>

The authors have nothing to disclose.

The first critical step in this protocol is isolating the mature eggs without damage. This can be achieved by gentile maneuvering of the eggs with the dissecting probe. Practice will enable this manipulation to be executed without damage to the eggs. A second crucial step is avoiding the loss of the sample when activation buffer is applied to the mature egg in oil. Application of the activation buffer should be done slowly and without contact with the coverslip. This step can be challenging if the microscope set-up does not allow for an easy access to the sample. Moving axillary parts of the microscope, such as a temperature incubator, is advisable. 
Modifications to this protocol can be made in order to visualize other fluorescently tagged factors, instead of Ca2+ at egg activation. More generally, different stages of Drosophila development or the results of adding a different buffer could be analyzed using this protocol. 
Troubleshooting may be required if the settings on the microscope are not optimal for visualizing the signal from the sample. This can be achieved by increasing or decreasing the laser power, altering capture range and adjusting the Z-stack parameters. Another issue might be eggs consistently moving out of the field of view upon the addition of activation buffer. If this happens regularly, allow the mature eggs to settle on the coverslips for a longer period of time, 15 to 20 minutes. Beware that using a higher viscosity oil will alter the displacement of the oil by activation buffer. 
This technique presented here is limited by the working distance of the objective, dehydration of the mature eggs and the ability to dissect the mature eggs without damage. However, when compared to in vivo imaging methods where the mature oocyte passes through the adult female and is deposited 13, our method enables more spatial resolution, the option to physically manipulate the mature egg before activation and the ability to test the role of egg activation without fertilization. 
There are many potential future applications of this technique, including testing reporter constructs for cellular components at egg activation and visualizing the Ca2+ wave in mutant backgrounds 14,24. Together with experimental and genetic tools, the ex vivo egg activation assay presented here enables the study of the trigger, propagation and downstream effects of the universally conserved Ca2+ wave.

A change in intracellular Ca2+ concentration at egg (oocyte) activation is a conserved component of all studied organisms 1,2. This event initiates a wide range of Ca2+-dependent processes, including resumption of the cell cycle and translation of stored mRNAs. Due to this requirement for Ca2+, visualizing the transient changes in intracellular Ca2+ concentrations has been of key interest.
Historically, model organisms were selected for studies on egg activation based on the size and availability of their eggs. Various visualization approaches have been utilized to follow and quantify changes in intracellular Ca2+ concentrations in these systems, including: the photoprotein aequorin in medaka fish 3; Ca2+ sensitive fluorescent dyes such as Fura-2 in sea urchin and hamster 4,5; and calcium green-1-dextran in Xenopus 6.
The generation and improvement of genetically encoded calcium indicators (GECIs) has transformed the ability to visualize Ca2+ dynamics in vivo 7. These genetic constructs are expressed in specific tissues and limit the need for invasive tissue preparations 8.
GCaMPs are a green fluorescent protein (GFP)-based class of GECIs that have been very effective due to their high Ca2+ affinity, signal-to-noise ratio and capacity to be customized 9-11. In the presence of Ca2+, the GCaMP complex undergoes a series of conformational changes, starting with the binding of Ca2+ to calmodulin, that results in an increased fluorescent intensity of the GFP component 9.
GCaMPs have been extensively used in research on Drosophila neurons to visualize changes in intracellular Calcium12. The recent application of GCaMP technology to visualize Ca2+ in mature Drosophila eggs has revealed a single transient Ca2+ wave at egg activation 13,14. The Ca2+ wave can be visualized at low magnification during ovulation in vivo 13 or at higher magnification using an ex vivo activation assay 13,14. In the ex vivo assay, individual mature oocytes are isolated from the ovaries and activated using a hypotonic solution, referred to as activation buffer, which has been shown to recapitulate the events of in vivo activation 15-17.
This ex vivo assay enables easy high resolution visualization of the Ca2+ wave under different experimental conditions including pharmacological disruption, physical manipulations and genetic mutants. This article demonstrates the preparation of mature Drosophila eggs for ex vivo activation and the subsequent microscopy used to visualize the Ca2+ wave using GCaMP. This approach can be used to test the initiation and control of the Ca2+ wave and to probe downstream outcomes.

<table><tbody><tr><td>Dry active yeast</td><td>Fleischman&amp;rsquo;s</td><td>#2192</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>MZ6</td><td>Various will work</td></tr><tr><td>Lamp</td><td>Mircotec Fibre optic</td><td>MF0-90</td><td>Various will work</td></tr><tr><td>Medical wipes</td><td>Kimberly-Clark Corporation</td><td>KLEW3020&amp;nbsp;</td><td></td></tr><tr><td>Marker pen</td><td>Stabilo</td><td>841/46</td><td>OHPen universal, black - available in most stationary shops. Superfine Width 0.4 mm</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pad</td><td>&amp;nbsp;Genesee Scientific</td><td>59-114 (789060 Dutscher)</td><td></td></tr><tr><td>Cover slip No. 1.5, 22 x 50 mm</td><td>International- No1-VWR Cat=631-0137-22-50mm</td><td>Menzel-Glaser-22-50mm-MNJ 400-070T&amp;nbsp;</td><td></td></tr><tr><td>Halocarbon oil, series 95</td><td>Halocarbon Products Corp.</td><td>Distributor in Europe:</td><td></td></tr><tr><td>Solvadis (GMBH)</td><td></td><td></td><td></td></tr><tr><td>Oil 10 S</td><td>VWR Chemicals</td><td>24627.188</td><td>Can be used instead of Halocarbon oil, series 95</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>Whilst these tool should be clean, use of an autoclave is not necessary, ethanol would be sufficient.</td></tr><tr><td>Probe</td><td>Fine Science Tools</td><td>10140-01</td><td></td></tr><tr><td>Glass pipette</td><td>Fisherbrand,</td><td>FB50251</td><td>Pasteur Pipettes, glass, unplugged, 150 mm length&amp;nbsp;</td></tr><tr><td>Vial</td><td>Regina Industries</td><td>P1014</td><td>GPPS&amp;nbsp; 80 mm x 25 mm</td></tr></tbody></table>

imaging calcium in drosophila at egg activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Here we have demonstrated how to prepare mature Drosophila eggs for ex vivo activation. Eggs expressing a GECI enable imaging of Ca2+ dynamics at egg activation and the beginning of embryonic development (Figure 1). It should be noted that depending on the GCaMP used, specifically the presence of a myristoyl group, results may have slight qualitative differences13 14. We have also demonstrated a role for functional actin during egg activation by the addition of an inhibitor of actin polymerization, cytochalasin D, to the activation buffer (Figure 2) 14. 
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A requirement for the cytoskeleton at egg activation is conserved. In C. elegans, following fertilization, cytoskeletal components are reorganized to prepare the one-cell embryo for the first asymmetrical division 21,22. In sea urchin, disruption of cytoskeletal re-organization following activation has been shown to affect the cell cycle by preventing contractile ring formation 23. The role of actin in mediating a Ca2+ wave and its downstream function at egg activation in Drosophila remains to be fully elucidated. 
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In Drosophila, co-visualization of the Ca2+ wave and actin cytoskeleton can be achieved using this ex vivo activation assay. Time series of multiple channels can be acquired and dynamics of the intracellular Ca2+ can be matched with changes in actin organization in the oocyte (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/54311/54311fig1.jpg" /> 
Figure 1: A Single Ca2+ Wave at Drosophila Egg Activation. Time series of ex vivo mature Drosophila egg expressing UASt-myrGCaMP5 following the addition of activation buffer (A-H). The rise in cytoplasmic Ca2+ originates at the posterior pole (B) and propagates (B-F) with an average velocity of approximately 1.5 &#181;m/sec. Initiation of the wave is typically observed within 3 min of the addition of activation buffer. Following activation, the intracellular calcium levels of the mature egg returns to pre-activation levels (G,H). See supplemental&#160;movie 1 (total time 33 min). Scale bars = 50 &#181;m. Max projection = 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54311/54311fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 2" src="/files/ftp_upload/54311/54311fig2.jpg" /> 
Figure 2: Addition of Cytochalasin D to Activation Buffer Perturbs Ca2+ Wave Propagation. Time series of ex vivo mature Drosophila egg expressing UASt-myrGCaMP5 following addition of activation buffer containing 10 &#181;g/ml cytochalasin D final concentration (A-H). Whilst the Ca2+ wave is initiated at the posterior pole (A), it is compromised and does not reach the anterior of the egg (F) (white arrowheads denote the front of the wave). No further Ca2+ changes were observed over 30 min). See supplemental movie 2 (total time 30 min). Scale bars = 50 &#181;m. Max projection = 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54311/54311fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54311/54311fig3.jpg" /> 
Figure 3: Co-visualization of Actin and Ca2+ at Egg Activation in Drosophila. Time series of ex vivo mature Drosophila egg expressing UASt-myrGCaMP5 (Cyan) and UASp-F-Tractin.tdTomato (Magenta) following addition of activation buffer (A-H). The Ca2+ wave initiates from the posterior pole and recovers as in Figure 1. Actin appears to be changing over time, white arrowheads (A vs H) . The lack of Ca2+ signal detection in the center of the mature egg is due to movement of the sample during image capture. Scale bars = 50 &#181;m. Max projection = 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54311/54311fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

imaging-calcium-in-drosophila-at-egg-activation

Research

JoVE Journal

Developmental Biology

7.8K Views.  University of Cambridge.  Dit artikel beschrijft een aanpasbare ex vivo protocol voor het visualiseren van Ca 2+ tijdens ei activering in Drosophila. 

Video: Imaging Calcium in Drosophila at Egg Activation

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Neuroscience

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Calcium Imaging in Individual Neurons Regulating Egg-Laying Behavior in Caenorhabditis elegans

Source: Ravi, B. et al., <a href="https://app.jove.com/v/56911">Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for calcium imaging in individual neurons of behaving Caenorhabditis elegans using genetically encoded fluorescent reporter proteins. It outlines the steps involved in imaging worm movement and measuring reporter fluorescence ratios from neurons to correlate intracellular calcium levels with neuronal activity and behavioral changes during egg-laying.

1. Strains, Culture Media, and Mounting of Animals

	Grow&#160;C. elegans&#160;worms at 20 &#176;C on standard 60-mm Nematode Growth Medium (NGM) agar ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Bioluminescence Imaging for Studying Calcium Transients in Drosophila Brain Structures

Source:&#160;Lark, A. R.,&#160;et. al.,&#160; In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.&#160;J. ...

Neuroimaging

Synaptic & Cellular Imaging

Drosophila In Vivo Calcium Imaging: A Method for Functional Imaging of Neuronal Activity

Source: Hancock, C. E., et al. <a href="https://www.jove.com/video/60288/" target="_blank">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes in vivo calcium imaging in Drosophila neurons using GCaMP. The featured protocol clip shows how to record changes in GCaMP fluorescence from mushroom body neurons during an olfactory conditioning experiment.

Source: Hancock, C. E., et al. In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster. J. Vis. Exp. (2019).
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Imaging Calcium in Drosophila at Egg Activation

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