We would like to thank Dr. Jeremy Smyth for imaging support and Ann C. Shenk for illustrations, production and videography. This work was supported by the National Institutes of Health (1R01GM127938 to R.T.C.).

1. Preparation of dissection and imaging media
NOTE: The media best suited for this live imaging experiment contains Schneider&#39;s Drosophila media containing 15% heat inactivated fetal bovine serum, 0.6x Pen-Strep, and 200 &#181;g/mL bovine insulin, hereafter referred to as Complete Schneider&#39;s media.
<ol>
	<li>Perform the media preparation under sterile conditions to ensure that it does not become contaminated. The media was developed to support Drosophila ovarioles for extended periods of time9.</li>
	<li>Add 15% heat inactivated fetal bovine serum, 0.6x Pen-Strep, and 200 &#181;g/mL bovine insulin to the Schneider&#39;s media.</li>
	<li>Mix the contents well and store at 4 &#176;C overnight. 
	NOTE: Insulin does not dissolve completely in the Complete Schneider&#39;s media, and you will notice a precipitate settle in the bottom of the tube.</li>
	<li>Make aliquots of the media, being sure to leave the precipitate as it will interfere with imaging. 
	&#8203;NOTE: This solution may be used within one month if stored in aliquots at 4 &#176;C.</li>
</ol>
2. Collection of Drosophila for dissection
NOTE: Detailed Drosophila collection and dissection procedures may also be found in Weil et al.10 and Parker et al.11.
<ol>
	<li>For optimal female germ cell imaging, first prepare a vial containing standard cornmeal fly food and a dab of wet yeast paste that is the consistency of peanut butter. This ensures the female flies are well-fed and will produce all follicle developmental stages for imaging.</li>
	<li>For optimally healthy flies, collect 0-1 day old females and transfer with males into a fly food vial containing wet yeast paste. 
	NOTE: Make sure the sleeping flies do not contact the yeast paste as they can stick to it.</li>
	<li>Feed the flies 3-7 days, changing the vial and the yeast paste daily. 
	&#8203;NOTE: Make sure the yeast paste contacts the fly food so it does not dry out.</li>
</ol>
3. Drosophila ovary dissection
NOTE: It is important to prepare the media solutions fresh because hydrogen peroxide is susceptible to oxidation and TMRE degrades over time.
<ol>
	<li>Right before dissection, in Complete Schneider&#39;s media, prepare a fresh aliquot of 2 &#181;M H2O2 solution, a fresh aliquot of 46 nM tetramethylrhodamine, ethyl ester (TMRE), and a fresh aliquot of a 46 nM TMRE solution containing 2 &#181;M H2O2.</li>
	<li>For ovary dissection, use two pairs of fine forceps and a pair of electrolytically sharpened tungsten needles12. To dissect the ovaries, fill 2-3 wells of a glass bottom dissecting dish (watch glass) with Complete Schneider&#39;s that has been warmed to room temperature.</li>
	<li>Anaesthetize the vial of fattened flies with carbon dioxide and segregate the desired number of female flies to be dissected. Place a single fly in the media using forceps.</li>
	<li>Under a dissecting microscope, gently grasp the fly by the thorax using one pair of fine forceps. With the other pair of forceps, grasp the posterior, and gently pull to remove the ovaries. 
	NOTE: if the ovaries do not come out smoothly using this method, the entire abdomen may also be removed from the fly, and the ovaries can be gently squeezed out of either end of the abdomen using forceps.</li>
	<li>Remove any extraneous cuticle or tissue, then transfer the ovaries to a new well containing fresh media. The ovaries should still be moving from the surrounding muscle sheath.</li>
</ol>
4. Preparing ovarioles for imaging
<ol>
	<li>Using sharpened tungsten needles, gently tease the ovarioles apart, taking care to remove the surrounding muscle sheath (Figure 1).</li>
	<li>Gently tease away any muscle sheath and nerve fibers attached to the isolated ovarioles (Figure 2). 
	NOTE: If the muscle sheath is not removed, the ovariole will twitch and move, causing problems with image acquisition (Video 1).</li>
	<li>If the subcellular structures of interest are endogenously labeled, proceed to Step 4.4. If the structures of interest will be labeled with a fluorescent dye, proceed to Section 5.</li>
	<li>Once the ovarioles have been cleanly dissected, using a micropipette, transfer them in a 100 &#181;L drop of Complete Schneider&#39;s imaging media into the glass depression of a glass bottom dish. The individual ovarioles will sink to the bottom of the droplet.</li>
	<li>Proceed to Section 6 for imaging. 
	&#8203;NOTE: For imaging mitochondria and Clu bliss particles, no more than five-ten minutes should elapse from the start of dissection to imaging.</li>
</ol>
5. Staining mitochondria with TMRE
NOTE: Additional detailed procedures on live staining of mitochondria with fluorescent dyes may be found in Parker et al. 2017.
<ol>
	<li>After step 4.3, transfer the isolated ovarioles in a 100 &#181;L drop of 46 nM TMRE media into the glass depression of a glass bottom dish. The individual ovarioles will sink to the bottom of the droplet.</li>
	<li>Incubate at room temperature for 20 minutes. Place the lid onto the glass-bottom dish and place the dish into a covered box for the duration of the experiment to protect from light. 
	NOTE: After incubation, samples can be imaged directly with no washes.</li>
	<li>Repeat steps 5.1-5.2 to prepare at least two dishes of TMRE-labeled ovarioles, one to serve as an experimental group and one to serve as a control.</li>
</ol>
6. Live image acquisition
<ol>
	<li>Once the ovarioles are mounted, place one of the glass bottom dishes onto the microscope and configure the imaging parameters as necessary. The optimum excitation/emission wavelengths for the TMRE used here are 549 nm/574 nm.</li>
	<li>After locating the desired field of view, acquire still images or brief videos of one or more ovarioles as desired as a record of pre-treatment conditions.</li>
</ol>
7. Addition of hydrogen peroxide during imaging
<ol>
	<li>Pause live imaging, remove the lid from the glass bottom dish, and carefully add 100 &#181;L of 2 &#181;M H2O2 solution to the dish using a micropipette if imaging endogenously labeled structures. If imaging mitochondria, carefully add 100 &#181;L of 46 nM TMRE with 2 &#181;M H2O2 solution to the dish using a micropipette (Video 2).
	<ol>
		<li>Avoid breaking the surface of the existing media droplet or adding the solution too quickly so as not to displace the ovarioles resting on the bottom of the dish (Video 2).</li>
	</ol>
	</li>
	<li>Replace the dish lid (dish cover), relocate and refocus the desired field of view if necessary, and resume imaging (Video 3, Video 4, Figure 3, Figure 4).
	<ol>
		<li>Take care to resume imaging as quickly as possible after H2O2 addition as the experimental treatment is time-sensitive (Video 5).</li>
	</ol>
	</li>
	<li>Acquire still images or brief videos of one or more ovarioles as desired. This will serve as a record of post-treatment conditions.</li>
	<li>For the control, place the second glass bottom dish onto the microscope and repeat Section 6.</li>
	<li>Repeat step 7.1, this time adding 100 &#181;L of TMRE-only media to the dish. If imaging endogenously labeled structures, add 100 &#181;L of the Complete Schneider&#39;s media-only solution.</li>
	<li>Repeat steps 7.2 and 7.3 to acquire data for the control group. 
	NOTE: For imaging mitochondria and Clu bliss particles, no more than three-five minutes should elapse from the addition of hydrogen peroxide to imaging.</li>
</ol>

<ol>
	<li>Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+iron+and+hydrogen+peroxide:+the+Fenton+reaction.">Toxicity of iron and hydrogen peroxide: the Fenton reaction.</a> Toxicology Letters. 82-83, 969-974 (1995).</li><li>Cox, R. T., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Balbiani+body+and+the+fusome+mediate+mitochondrial+inheritance+during+Drosophila+oogenesis.">A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis.</a> Development. 130 (8), 1579-1590 (2003).</li><li>Cox, R. T., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clueless,+a+conserved+Drosophila+gene+required+for+mitochondrial+subcellular+localization,+interacts+genetically+with+parkin.">Clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin.</a> Disease Models &amp; Mechanisms. 2 (9-10), 490-499 (2009).</li><li>Sen, A., Kalvakuri, S., Bodmer, R., Cox, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clueless,+a+protein+required+for+mitochondrial+function,+interacts+with+the+PINK1-Parkin+complex+in+Drosophila.">Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila.</a> Disease Models &amp; Mechanisms. 8 (6), 577-589 (2015).</li><li>Sheard, K. M., Thibault-Sennett, S. A., Sen, A., Shewmaker, F., Cox, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clueless+forms+dynamic,+insulin-responsive+bliss+particles+sensitive+to+stress.">Clueless forms dynamic, insulin-responsive bliss particles sensitive to stress.</a> Developmental Biology. 459 (2), 149-160 (2020).</li><li>Sen, A., Cox, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clueless+is+a+conserved+ribonucleoprotein+that+binds+the+ribosome+at+the+mitochondrial+outer+membrane.">Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane.</a> Biology Open. 5 (2), 195-203 (2016).</li><li>Parton, R. M., Valles, A. M., Dobbie, I. 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=Isolation+of+Drosophila+egg+chambers+for+imaging.">Isolation of Drosophila egg chambers for imaging.</a> Cold Spring Harbor Protocols. 2010 (4), 5402 (2010).</li><li>Morris, L. X., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live+imaging+provides+new+insight+into+stem+cell+regulation+and+germline-soma+coordination+in+the+Drosophila+ovary.">Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary.</a> Development. 138 (11), 2207-2215 (2011).</li><li>Prasad, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+border+cell+migration+analyzed+using+time-lapse+live-cell+imaging.">Cellular and molecular mechanisms of border cell migration analyzed using time-lapse live-cell imaging.</a> Developmental Cell. 12 (6), 997-1005 (2007).</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> Journal of Visualized Experiments. (60), e3679 (2012).</li><li>Parker, D. J., Moran, A., Mitra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+Mitochondrial+Structure+and+Function+in+Drosophila+Ovaries.">Studying Mitochondrial Structure and Function in Drosophila Ovaries.</a> Journal of Visualized Experiments. (119), e54989 (2017).</li><li>Brady, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+making+fine,+durable+dissecting+needles+by+sharpening+tungsten+wire+electrolytically.">A simple technique for making fine, durable dissecting needles by sharpening tungsten wire electrolytically.</a> Bulletin of the World Health Organization. 32 (1), 143-144 (1965).</li><li>Hudson, A. M., Petrella, L. N., Tanaka, A. J., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mononuclear+muscle+cells+in+Drosophila+ovaries+revealed+by+GFP+protein+traps.">Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps.</a> Developmental Biology. 314 (2), 329-340 (2008).</li><li>Wang, S., Hazelrigg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+for+bcd+mRNA+localization+from+spatial+distribution+of+exu+protein+in+Drosophila+oogenesis.">Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis.</a> Nature. 369 (6479), 400-403 (1994).</li></ol>

The authors have nothing to disclose.

This protocol could be a useful addition as a control for artifacts due to ovary dissection and tissue incubation for any live imaging experiment. The critical steps are similar to those found for other live imaging protocols. Learning how to dissect whole Drosophila ovaries takes practice; however, this skill can usually be learned fairly quickly with the appropriate dissection tools. More difficult to master is removing the muscle enveloping the ovaries and each ovariole13. This must be done to ensure muscle contractions do not interfere with image acquisition. If using sharpened tungsten needles to do this does not prove successful, the germarium at the tip of the ovariole can be grasped with forceps and the ovariole pulled from the muscle sheath. However, this technique is problematic if the earliest developmental stages are to be examined because they can become damaged. Another key step is to not dislodge the ovarioles resting on the bottom of the dish when adding H2O2. An additional important aspect is shared by all live imaging: the researcher should ensure that the structure of interest is fluorescently well-labeled before treatment. The dishes used here (Table of Materials) are commonly used for live imaging; however, any dish or slide with a glass coverslip on the bottom or even a large glass coverslip should work as long as the drop of media can be covered to prevent media evaporation. While we use a particular microscope, any inverted microscope with an objective of sufficient magnification to see the subcellular structure in question and an attached camera that has sufficient resolution and image capture rate should work.
While our laboratory is primarily interested in mitochondrial function, this method could be helpful examining the dynamics and localization of any subcellular structure or organelle, such as the nucleus, cytoskeleton or endoplasmic reticulum. However, this method has limitations. In order to add hydrogen peroxide, the tissue must be in an aqueous media. An alternative method for live imaging is to use halocarbon oil, which has been instrumental in describing many important processes in Drosophila ovarioles including the first example of dynamic movement of GFP in a model organism7,14. In addition, adding hydrogen peroxide to the media causes wide-spread oxidative damage which may be too general an insult to the tissue to be informative for the cellular process of interest, particularly for longer experiments examining development. Though it may not be feasible to perform experiments which require visualizing the cell over long periods of time due to this rapid, extensive, and likely irreversible oxidative damage, we have seen that the acute hydrogen peroxide treatment we have described is applicable to most stages of oogenesis as we are able to see the same effects in most stages within the imaging time period. Given the low cost and ease of the protocol, it may be a useful control for damage and can be used as a treatment before fixation and antibody labeling as well.
In our hands, H2O2 treatment mimics the changes in mitochondrial mislocalization and Clu bliss particle dispersion that we see in various Drosophila mutants. It also mimics results we see for new researchers in the lab learning dissection techniques. Therefore, this method clearly revealed that sample preparation and general cellular stress can lead to unexpected and previously unexplained changes to mitochondrial mislocalization and the presence of bliss particles. Moving this technique forward, hydrogen peroxide concentrations could be modulated using a higher or lower concentration. If a cellular effect is seen using a lower concentration, it is possible the stress phenotype may be reversible by replacing the media with Complete Schneider's. Different cell stressors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), arsenite or simple heat shock might prove useful for general cellular stress for other subcellular structures. Since live imaging of ex vivo tissues requires manual manipulation and incubation in different media, this control should be a useful addition to ensure any observations are as close to normal physiology as possible.

Multiple different cellular stressors may arise during the experimental culture and manipulation of tissues ex vivo, including heat shock, oxidative stress, osmotic stress, nutritional stress, and toxicity conditions. Live imaging is a powerful tool used to visualize real-time changes in ex vivo tissues after experimental treatment and manipulation. Fine tissue dissections and manipulation take practice, and the amount of time from dissection to imaging can vary depending on experience. The rationale for developing this method is based on the concern that preparing tissue for live imaging can cause cellular stress during dissection and imaging preparation. This could be particularly problematic for processes sensitive to changes in cellular metabolism and available oxygen levels, such as mitochondrial function. While having a parallel wild type sample is an important control, there is still the possibility that some or all observed changes in subcellular structures could be due to damage or cell stress from dissection and do not reflect normal physiology or the treatment or mutation being studied.
To address this potential problem, we use hydrogen peroxide addition during live imaging in order to induce cellular oxidative damage1. The purpose of this method is to induce damage to tissues in order to monitor the effect on subcellular structures. This protocol is useful for two purposes: 1) determining whether changes in subcellular localization of the structure of interest is due to the stress caused by inexperienced dissection and 2) once the researcher is confident with the dissection techniques described to monitor the effect of controlled stress on the structure of interest. Here we show two examples of how increased oxidative damage causes changes in two subcellular structures, mitochondria and Clu bliss particles. To do this, we use the Drosophila ovary which is a common model for live imaging studies. The first example examines mitochondrial localization. In our experience, normal mitochondrial localization in female germ cells is highly sensitive to perturbations and can act as a harbinger of cellular stress. Mitochondria in Drosophila female germ cells are normally evenly dispersed throughout the cytoplasm2. Hydrogen peroxide addition causes the organelles to quickly mislocalize and cluster in a similar manner to various mutations3,4,5. The second example are bliss particles formed by Clueless (Clu). Clu is a ribonucleoprotein that is diffuse throughout the cytoplasm; however, it also forms mitochondria-associated particles under optimal cellular conditions5. Because the presence of Clu particles is dependent upon healthy cellular conditions, we have termed them &#34;bliss&#34; particles3,5,6. Addition of hydrogen peroxide causes these particles to quickly disperse and become homogeneous in the cytoplasm5. In the course of our studies, we have observed changes in localization of both of these subcellular structures, but only after performing live imaging studies could we fully appreciate the effect of cellular stress and oxidative damage on localization and dynamics of mitochondria and bliss particles.
The utility of this protocol as an addition to already established or alternative methods depends on several factors. First, the imaging protocol must be amenable to drug-addition. If the sample is mounted under a coverslip and in halocarbon oil, this method would not be possible7. H2O2 addition causes a rapid rise in oxidative damage, therefore, this timescale may not be appropriate. Oxidative damage may be regarded as a proxy for hypoxia; however, it may be too harsh or too generalized to function as an appropriate control for damage for certain subcellular components. Finally, for imaging experiments that last hours such as those following a developmental process, H2O2 addition may be too strong (for example8). Testing a concentration curve may overcome this limitation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Active dry yeast</td>
			<td>Red Star&#174;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 gas</td>
			<td></td>
			<td></td>
			<td>99.9% purity</td>
		</tr>
		<tr>
			<td>CO2 pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope, Nikon SMZ645 model</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needles - PrecisionGlide needles</td>
			<td>BD</td>
			<td>305165</td>
			<td>B-D 21G1 size</td>
		</tr>
		<tr>
			<td>Dissecting needles - PrecisionGlide syringes</td>
			<td>BD</td>
			<td>309657</td>
			<td>Luer-Lok tip, 3 mL size</td>
		</tr>
		<tr>
			<td>Dissecting needles - tungsten wire</td>
			<td>Electron Microscopy Sciences</td>
			<td>73800</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (2 pairs)</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>NI-150 High Intensity Illuminator</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum, Heat Inactivated</td>
			<td>Fisher Scientific</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Schneider&#39;s Drosophila Media</td>
			<td>Sigma-Aldrich</td>
			<td>21720-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution, 30% (w/w) in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Equivalent scopes may also be used</td>
		</tr>
		<tr>
			<td>Lonza BioWhittaker Antibiotics: Penicillin-Streptomycin mixtures</td>
			<td>Fisher Scientific</td>
			<td>17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>MatTek Corporation Glass Bottom Dishes, 35 mm</td>
			<td>Fisher Scientific</td>
			<td>&#160;NC9344527</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes and tips of appropiate size</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 1.7 mL</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)</td>
			<td>AnaSpec</td>
			<td>AS-88061</td>
			<td></td>
		</tr>
		<tr>
			<td>w[1118]</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>5905</td>
			<td>Wild-type flies</td>
		</tr>
		<tr>
			<td>y w; clu[CA06604]</td>
			<td></td>
			<td>Available upon request.</td>
			<td>Clu::GFP trap flies</td>
		</tr>
	</tbody>
</table>

visualizing the effects of oxidative damage on drosophila egg chambers using live imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The described protocols can be used to study the effects of hydrogen peroxide during live imaging of Drosophila ovaries. As shown in Figure 3, Figure 4, Video 3 and Video 4, this procedure provides an effective means to visualize tissue changes and dynamics after experimental treatment in real-time. Importantly, this protocol is specific for the addition of H2O2 to ovarioles&#160;while imaging; however, it may be adapted for the exogenous addition of other drugs or reagents of interest. In addition, follicles may be labeled with any fluorescent dyes of interest (e.g., tetramethylrhodamine (TMRE), LysoTracker) prior to imaging (Video 3). The most critical steps to obtaining clear imaging results are 1) the proper dissection and isolation of single ovarioles with all contractile elements removed (Figure 1 and Figure 2) and 2) the speed at which imaging is restarted after hydrogen peroxide addition. Video 4 is an illustration of a properly dissected follicle that remains steady throughout the imaging duration as compared to Video 1, which illustrates a poorly dissected follicle exiting the field of view during imaging. Video 5 is an illustration in which the H2O2 effects on TMRE-labeled mitochondria&#160;have already progressed prior to the start of imaging as a result of too much elapsed time. As compared to Video 3 in which imaging was restarted immediately after hydrogen peroxide addition (time 0) and intact, dispersed mitochondria are still visible, the mitochondria in Video 5 have already began to visibly clump and lose their membrane potential upon the restart of imaging. This issue is mostly attributed to disruption of the sample positions during H2O2 addition and can be alleviated by following the technique to keep the imaging media and sample position intact (Video 2). Of note, in control experiments performed without H2O2 added to the media, the mitochondria in follicles remains properly dispersed, and the TMRE dye remains sequestered in the mitochondria.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62157/62157fig01.jpg" /> 
Figure 1:&#160;Isolation of single ovarioles from Drosophila ovaries. (A) Cartoon indicating a pair of ovaries, a single ovariole (arrow) with the germarium at the tip (arrowhead) and the two muscle sheaths that surround the ovariole (brown, epithelial) and the ovary (brown, peritoneal). (B) Dissected Drosophila ovary. (C) Subsequent separation of the teased ovary into individual ovarioles (arrow). Scale bar = 100 um. <a href="https://www.jove.com/files/ftp_upload/62157/62157fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62157/62157fig02.jpg" /> 
Figure 2: Removal of nerve fibers and contractile elements from single ovarioles. (A) Single ovariole with remnants of nerve tissue and the muscle sheath still attached (arrow). (B) Gentle removal of all remaining tissue attached to ovariole in A (arrow). Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62157/62157fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62157/62157fig03.jpg" /> 
Figure 3:&#160;H2O2 causes mitochondrial mislocalization. (A-A&#8243;) Live-image stills of a well-fed cluCA06604 (Clu::GFP flies)&#160;follicle. Addition of H2O2 causes mitochondria to clump over the duration of imaging. TMRE labeling of mitochondria indicates that mitochondria are initially dispersed at time 0 (A), and that mitochondria start to clump after H2O2 addition (A&#8242;) At a later time-point, the TMRE labeling becomes spotty due to mitochondria losing their membrane potential and therefore their ability to sequester the dye (A&#8243;). White = TMRE (A-A&#8243;). Scale bars: 10 &#181;m in (A) for A-A&#8243;5. <a href="https://www.jove.com/files/ftp_upload/62157/62157fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62157/62157fig04.jpg" /> 
Figure 4:&#160;H2O2 disperses Clu. (A-A&#8243;) Live-image stills of a well-fed cluCA06604 follicle. Addition of H2O2 causes particles to disperse and become homogeneous in the cytoplasm over the duration of imaging. White = Clu::GFP (A-A&#8243;). Scale bar: 40 &#181;m in (A) for A-A&#8243;5. <a href="https://www.jove.com/files/ftp_upload/62157/62157fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Follicle from a cluCA06604 female. As described in Figure 2, failure to remove nerve fiber contractile elements from single ovarioles will cause marked drifting and movement of the ovarioles during imaging and subsequent inability to analyze imaging data. White = Clu::GFP.&#160;<a href="https://www.jove.com/files/ftp_upload/62157/Video 1 - Failure to remove contractile elements.mov" target="_blank">Please click here to download this Video.</a>
Video 2: Proper addition of H2O2 to sample dish. Hydrogen peroxide should be added to the sample dish using an appropriately sized micropipette. Dispensing H2O2 without breaking media surface. Care is taken to avoid breaking the surface of the imaging media to minimize sample drift during hydrogen peroxide addition.&#160;<a href="https://www.jove.com/files/ftp_upload/62157/Video 2 - Proper Addition of H2O2 to Sample Dish.mp4" target="_blank">Please click here to download this Video.</a>
Video 3: H2O2 addition during imaging of TMRE labeled follicles. Follicle from a cluCA06604 female stained with TMRE. H2O2 causes mitochondrial mislocalization in Drosophila follicles. White = TMRE dye. Imaged one frame per 15 s for 20 min, and the video is 10 frames per second5.&#160;<a href="https://www.jove.com/files/ftp_upload/62157/Video 3 - H2O2 stresses mitochondria.avi" target="_blank">Please click here to download this Video.</a>
Video 4: H2O2 addition during imaging of Clu::GFP follicles. Follicle from a cluCA06604 female. H2O2 disperses Clu particles as described in Figure 4. White = Clu::GFP. Imaged one frame per 15 s for 15 min, and the video is 10 frames per second.5&#160;<a href="https://www.jove.com/files/ftp_upload/62157/Video 4 - H2O2 disperses particles.avi" target="_blank">Please click here to download this Video.</a>
Video 5: Delayed imaging after H2O2 addition. Follicle from a cluCA06604 female. Addition of hydrogen-peroxide to follicles is a time-sensitive treatment. The restart of imaging was delayed in this video as a result of sample displacement during H2O2 addition. The mitochondria have already begun to visibly clump and lose their membrane potential upon the restart of imaging at time 0 (as compared to time 0 in Video 3). Failure to resume imaging quickly after treatment will result in inaccurate and unusable results as the early experimental effects will be missed prior to imaging. White = TMRE. Imaged one frame per 15 s for 20 min, and the video is 10 frames per second.&#160;<a href="https://www.jove.com/files/ftp_upload/62157/Video 5 - Delayed imaging after H2O2 addition.mov" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging at JoVE.com

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

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3.5K Views.  Uniformed Services University. The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Video: Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

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 ...

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.

Imaging Calcium in Drosophila at Egg Activation

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

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

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...