We are grateful to Chun-lan Hsu and Yu-Chi Yang for preparing the fly food and maintaining the fly stocks, and to Su-Ping Lee and the IMB Imaging Core for their help with the confocal microscopy. This study was supported by grants to Y.H.S. (NSC 101-2321-B-001 -004, NSC 100-2321-B-001 -012, NSC 102-2321-B-001 -002, MOST 103-2311-B-001 -035 -MY3) from the National Science Council and the Ministry of Science and Technology of the Republic of China.

1. Pre-experimental Setup
<ol>
	<li>Collect a fly egg that expresses nuclear green fluorescent protein (GFP) under m&#948;0.5-Gal4 and culture at 25 &#176;C for 5 days.</li>
	<li>Microwave 0.7 g of low-melting agarose in 10 mL of 1x Phosphate-buffered Saline (PBS) until the agarose is completely dissolved. Keep the 0.7% low-melting gel in a 37 &#176;C incubator until use.</li>
	<li>Sterilize all equipment, including forceps, scissors, 42 mm x 0.17 mm coverslips, a 9-well glass spot plate, and a magnetic culture chamber, with 70% ethanol for more than 12 h.</li>
	<li>Prepare the culture medium3: Schneider&#39;s Drosophila medium supplemented with 2% Fetal Bovine Serum (FBS), 0.5% penicillin-streptomycin, and 1.25 mg/mL insulin. To avoid contamination, prepare the medium in a cell culture hood. Store the prepared culture medium at 4 &#176;C and used within a month.</li>
	<li>Clean the dissection platform and the experimenter&#39;s hands with 70% ethanol before starting the dissection.</li>
</ol>
2. Disc Dissection
<ol>
	<li>Air dry all the equipment from 70% ethanol.</li>
	<li>Select a third-instar larva from a fly vial. To avoid contamination from the fly vial, wash the larva in 1x PBS 3x to remove the waste.</li>
	<li>Dissect the cleaned larva in 1 mL of culture medium (at RT) in the 9-well glass spot plate under a dissection microscope.</li>
	<li>Grasp the larva with a pair of forceps at about one third of the way from the posterior end and another forceps at the mouth hook. Pull the two forceps in opposite directions to release the internal tissues.
	<ol>
		<li>Remove the salivary glands, fat body, and cuticle from the eye-brain complex attached to the mouth hook. If the ventral nerve cord is still attached, cut it away with scissors.</li>
	</ol>
	</li>
	<li>Use one pair of forceps to grasp the mouth hook and one pair of forceps to remove a piece of tissue that connects the brains and eye discs in the dorsal part. 
	NOTE: If this tissue is not removed, the two eye-antennal discs will be close to each other and the brain. The eye discs will then not lie flat on the coverslip because this tissue, the brain, and the eye-antennal disc will form a triangle.</li>
	<li>Rotate the whole complex to a ventral-upward position. Remove the filament that connects the hook or disc to the brain. 
	NOTE: If the filament is not removed, the eye-antennal disc will split into two piece when the mouth hook is removed.
	<ol>
		<li>Remove the mouth hook. Take care not to damage the eye disc during the process. 
		NOTE: The eye disc is now free in the medium and only connected to brain by the optical stalk.</li>
	</ol>
	</li>
</ol>
3. Mounting
<ol>
	<li>Air dry and assemble the components of the chamber with a 42 mm x 0.17 mm coverslip. Attach a double layer of O-rings to the center of the cover slip to hold the agarose.</li>
	<li>Assemble the chamber with coverslip. Put the 42 mm x 0.17 mm coverslip on the stage acceptor. Place the silicon O-ring on the cover slip. Place the base. Lock the sealing locker and place the cover (Figure 1).</li>
	<li>Carefully transfer the dissected eye-brain complex to the center of O-ring using a 20 &#181;L micropipette with a 10-200 &#181;L tip.</li>
	<li>Remove most of the medium and add 12 &#181;L of 0.7% low-melting agarose (at 37 &#176;C) to the sample. 
	NOTE: The position of the eye disc can be changed with forceps before the solidification of the agarose. The agarose will solidify within 5 min.
	<ol>
		<li>Add 1 mL of culture medium at room temperature to the center of O-ring; each O-ring can load up to 4 samples. Make sure that the agarose is glued to the O-ring to avoid the drifting of the sample. 
		NOTE: No aeration or circulation of the medium is necessary.</li>
	</ol>
	</li>
</ol>
4. Confocal Microscopy
<ol>
	<li>Place the chamber on the stage of an inverted confocal microscope for 30 min to equilibrate the temperature. This can prevent Z-axis drift during the imaging.</li>
	<li>Before long-term live imaging, check whether the tissue is intact by differential interference contrast microscopy.</li>
	<li>To avoid the phototoxicity, use a laser power below 2 mW. Use a 40X objective for imaging.</li>
	<li>Acquire 62 &#181;m Z-stack images in 12 min at an optical interval of 1 &#181;m. Acquire 40 cycles of scans over a total of 10 h. Ensure that each cycle contains 12 min of imaging and 3 min of resting.</li>
</ol>

<ol>
	<li>Cohen, S. M., Bate, M., Martinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+disc+development.">Imaginal disc development.</a> The Development of Drosophila melanogaster. 2, 747-842 (1993).</li><li>Auerbach, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+legs,+wings+and+halteres+in+wild+type+and+some+mutant+strains+of+Drosophila+melanogaster.">The development of the legs, wings and halteres in wild type and some mutant strains of Drosophila melanogaster.</a> Proc. R. Soc. Edinb. B. 58, 787-815 (1936).</li><li>Tsao, C. K., Ku, H. Y., Lee, Y. M., Huang, Y. F., Sun, Y. H. <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+Ex+Vivo+Culture+and+Live+Imaging+of+Drosophila+Larval+Imaginal+Discs.">Long-term Ex Vivo Culture and Live Imaging of Drosophila Larval Imaginal Discs.</a> PLoS One. 11 (9), e0163744 (2016).</li><li>Ready, D. F., Hanson, T. E., Benzer, 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+the+Drosophila+retina,+a+neurocrystalline+lattice.">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev Biol. 53 (2), 217-240 (1976).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye:+the+morphogenetic+furrow+and+the+second+mitotic+wave.">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113 (3), 841-850 (1991).</li><li>Tomlinson, A., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+differentiation+in+Drosophila+ommatidium.">Neuronal differentiation in Drosophila ommatidium.</a> Dev Biol. 120 (2), 366-376 (1987).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Development of Drosophila Melanogaster. , 1277-1316 (1993).</li><li>Adler, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+signaling+and+morphogenesis+in+Drosophila.">Planar signaling and morphogenesis in Drosophila.</a> Dev Cell. 2 (5), 525-535 (2002).</li><li>Choi, K. W., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+glia+along+photoreceptor+axons+in+the+developing+Drosophila+eye.">Migration of glia along photoreceptor axons in the developing Drosophila eye.</a> Neuron. 12 (2), 423-431 (1994).</li><li>Strutt, D. I., Mlodzik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ommatidial+polarity+in+the+Drosophila+eye+is+determined+by+the+direction+of+furrow+progression+and+local+interactions.">Ommatidial polarity in the Drosophila eye is determined by the direction of furrow progression and local interactions.</a> Development. 121 (12), 4247-4256 (1995).</li><li>Gaengel, K., Mlodzik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egfr+signaling+regulates+ommatidial+rotation+and+cell+motility+in+the+Drosophila+eye+via+MAPK/Pnt+signaling+and+the+Ras+effector+Canoe/AF6.">Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6.</a> Development. 130 (22), 5413-5423 (2003).</li><li>Li, C., Meinertzhagen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+for+the+primary+culture+of+eye+imaginal+discs+from+Drosophila+melanogaster.">Conditions for the primary culture of eye imaginal discs from Drosophila melanogaster.</a> J Neurobiol. 28 (3), 363-380 (1995).</li><li>Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+eye.">Pattern formation in the Drosophila eye.</a> Curr Opin Genet Dev. 17 (4), 309-313 (2007).</li><li>Treisman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+differentiation+in+Drosophila.">Retinal differentiation in Drosophila.</a> Wiley Interdiscip Rev Dev Biol. 2 (4), 545-557 (2013).</li><li>Huisken, J., Swoger, J., Del Bene, ., Wittbrodt, F., J, E. H., Stelzer, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy.">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305 (5686), 1007-1009 (2004).</li><li>Keller, P. J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vivo+imaging+of+entire+embryos+with+Digital+Scanned+Laser+Light+Sheet+Fluorescence+Microscopy.">Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy.</a> Curr Opin Neurobiol. 18 (6), 624-632 (2008).</li><li>Winter, P. W., Shroff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faster+fluorescence+microscopy:+advances+in+high+speed+biological+imaging.">Faster fluorescence microscopy: advances in high speed biological imaging.</a> Curr Opin Chem Biol. 20, 46-53 (2014).</li><li>Bosveld, F., 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=Mechanical+Control+of+Morphogenesis+by+Fat/Dachsous/Four-Jointed+Planar+Cell+Polarity+Pathway.">Mechanical Control of Morphogenesis by Fat/Dachsous/Four-Jointed Planar Cell Polarity Pathway.</a> Science. 336 (6082), 724-727 (2012).</li><li>Ghannad-Rezaie, M., Wang, X., Mishra, B., Collins, C., Chronis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Chips+for+In+Vivo+Imaging+of+Cellular+Responses+to+Neural+Injury+in+Drosophila+Larvae.">Microfluidic Chips for In Vivo Imaging of Cellular Responses to Neural Injury in Drosophila Larvae.</a> Plos One. 7 (1), (2012).</li><li>Heemskerk, I., Lecuit, T., LeGoff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clonal+analysis+based+on+chronic+in+vivo+imaging+allows+multiscale+quantification+of+growth+in+the+Drosophila+wing+disc.">Dynamic clonal analysis based on chronic in vivo imaging allows multiscale quantification of growth in the Drosophila wing disc.</a> Development. 141 (11), 2339-2348 (2014).</li><li>Taylor, J., Adler, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+rearrangement+and+cell+division+during+the+tissue+level+morphogenesis+of+evaginating+Drosophila+imaginal+discs.">Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs.</a> Dev Biol. 313 (2), 739-751 (2008).</li><li>Ward, R. E., Reid, P., Bashirullah, A., D'Avino, P. P., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+in+living+animals+reveals+dynamic+developmental+responses+to+ecdysone+during+drosophila+metamorphosis.">GFP in living animals reveals dynamic developmental responses to ecdysone during drosophila metamorphosis.</a> Dev Biol. 256 (2), 389-402 (2003).</li><li>Gibbs, S. M., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+cyclic+GMP+regulate+retinal+patterning+in+the+optic+lobe+of+Drosophila.">Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila.</a> Neuron. 20 (1), 83-93 (1998).</li><li>Fristrom, J. W., Logan, W. R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+and+Minimal+Culture+Requirements+for+Evagination+of+Imaginal+of+Drosophila-Melanogaster+in-Vitro.">Synthetic and Minimal Culture Requirements for Evagination of Imaginal of Drosophila-Melanogaster in-Vitro.</a> Dev Biol. 33 (2), 441-456 (1973).</li></ol>

The authors declare that they have no competing financial interests.

In this study, we provide a detailed protocol for a long-term live imaging experiment on Drosophila imaginal discs. We spent a lot of time practicing the careful dissection to avoid disc damage and to allow for the attachment of the disc close to the coverslips. We tried Poly-L-Lysine (PLL) and low-melting agarose to hold the tissue; in the end, the low-melting agarose showed a better ability to hold the tissue. Although only the eye disc was used in this paper to demonstrate the normal occurrence of photoreceptor differentiation and ommatidial rotation during the 10 h live imaging period, this method can also be applied to at least one other disc, the wing disc, as demonstrated in a previous study3. Moreover, the culture medium preparation and live imaging setup are simple and do not require aeration or medium circulation.
Continuous live imaging can provide a clear temporal sequence of biological events. In our earlier report of glia differentiation and migration in the eye disc, we provided direct proof that wrapping glia differentiate from existing glia after migrating to the anterior of the eye disc3. Long-term ex vivo culture allows for the specific laser-activated labelling of cells, such as the photo-conversion of the KAEDE fluorescent protein, to follow their behaviors3. It can also accommodate the testing of chemicals that are added directly to the culture medium; thus, it can be used as a drug screening platform. Since some dynamic process occur within minutes or seconds, high temporal resolution is required. To enhance the temporal resolution, light sheet microscopy15,16 or spinning disc microscopy17 may be a good option.
The current culture condition is not perfect. It does not support disc growth. Cell proliferation is only supported for up to 12 h3. After 12 h in ex vivo culture, the rate of photoreceptor differentiation starts to decrease3. This may be due to the lack of certain nutrients or hormones. Since the major difference in this culture medium is the high concentration of insulin, the mammalian insulin may be partially mimicking the function of endogenous insulin-like peptides. The addition of fly extract, the insect blood sugar trehalose, and various concentration of the molting hormone 20-hydroxyecdysone, were not beneficial3. However, the 12-18 h culture period is sufficient to study many developmental processes. Alternative culturing methods for culturing larval imaginal discs have been compared3, and our culturing and imaging condition provides the longest time window and most clarity for live imaging. Although discs within live larvae and pupae can be imaged directly18,19,20,21,22, the resolution and time window for observations are limited. Late larval and pupal discs can be cultured for a long time23,24, but the discs undergo significant morphogenesis. To take advantage of the flat, 2D larval discs, our culturing and imaging method is the best solution so far.

The Drosophila larval imaginal discs have been a favored experimental system for the study of a wide variety of biological mechanisms. These discs invaginate from the embryonic epithelium and become sac-like structures built up by two epithelial layers, Peripodial Epithelium (PE) and Disc Proper (DP)1,2. The imaginal disc cells proliferate and progressively differentiate during the larval and pupal stages and eventually develop into the adult body structures. Due to the flat and simple two-layer structure of the imaginal discs, they are easy to observe when dissected from larvae. However, despite several efforts by many groups, the current methods for the long-term culture of the imaginal discs have not been satisfactory. We have recently developed a culture method that can sustain the normal development of multiple imaginal discs for up to 18 h and that allows live imaging3. The culture method can support cell differentiation and migration, but it can support cell proliferation for only 7-12 h and cannot support disc growth. The major difference in the culture medium is the high concentration of insulin3. The method is simple, and no special aeration or medium circulation is required.
One of the best-studied imaginal discs is the eye-antennal disc. The Drosophila eye is built up of approximately 800 ommatidia. Each ommatidium is comprised of eight photoreceptor cells (R1-R8). In the larval eye disc, the photoreceptor cells differentiate following the passage of the Morphogenetic Furrow (MF), which sweeps across the eye disc from posterior to anterior4,5. The photoreceptors in an ommatidium differentiate in sequence, beginning with R8, followed by R2/R5, R3/R4, R1/R6, and R76. The ommatidia in the dorsal and ventral halves of the eye disc undergo a 90&#176; rotation in opposite directions, resulting in opposite chirality7,8. The rotation process occurs in two stages: first, a 45&#176; rotation begins at and is completed at the sixth ommatidia row behind the MF; the second 45&#176; rotation is completed at about row 169,10. This study used ommatidial rotation, marked by the R3/4-specific m&#948;0.5-Ga411, to demonstrate the normal cellular differentiation and dynamics that can be observed through this method to culture and live-image the eye disc.

<table><tbody><tr><td>Low-melting agarose</td><td>AMRESCO&amp;nbsp;</td><td>0815-25G</td><td></td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>AMRESCO</td><td>0780-2PK</td><td></td></tr><tr><td>42 mm x 0.17 mm cover slips&amp;nbsp;</td><td>PST</td><td>G401-42</td><td></td></tr><tr><td>Schneider&amp;rsquo;s &lt;em&gt;Drosophila&lt;/em&gt; medium&amp;nbsp;</td><td>Thermo</td><td>21720-024</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F9665</td><td></td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>GIBCO</td><td>759</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I9278</td><td></td></tr><tr><td>Culture chamber&amp;nbsp;</td><td>PECON</td><td></td><td>POC-R2 Cell Cultivation System</td></tr><tr><td>40X objective</td><td></td><td></td><td>C-Apochromat 40X/1.2W Korr objective&amp;nbsp;</td></tr><tr><td>Fly strain:nls.GFP</td><td>Bloominggton</td><td>4775</td><td></td></tr></tbody></table>

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.

In this example, the R3/R4 photoreceptors were labeled with m&#948;0.5-Ga4 to monitor photoreceptor differentiation. m&#948;0.5-Ga4 drives strong expression in R4 and weaker expression in R311, making it an excellent marker for R3 and R4, as well as for the process of ommatidial rotation. In Movie 1, R3 and R4 were labelled with GFP. At the beginning of the 10 h live-imaging session, there were 8 rows of ommatidial clusters (Figure 2A and Movie 1) and 14 rows of ommatidial clusters at the end (Figure 2B). The rate of ommatidial differentiation was 1.67 h per row, which is close to the rate of 1.5-2 h/row in in vivo discs 7,8,9,10,11,12,13,14. Based on the relative positions of R3 and R4, ommatidial rotation occurred in the ex vivo cultured disc during live imaging (Figure 2A&#39;, A&#39;&#39;, B&#39;, and B&#39;&#39;). A single R3/R4 cluster was labeled with a white sphere in the first frame of Movie 2. In the beginning, the R3/R4 axis appears to be perpendicular to the equator (Figure 2C and Movie 2). It then appears to rotate 15&#176;, 30&#176;, and 45&#176; in 3 (Figure 2C&#39;), 6 (Figure 2C&#39;&#39;), and 9 (Figure 2C&#39;&#39;&#39;) h, respectively. This data suggest that this method can sustain photoreceptor differentiation during the 10 h live imaging.
<img alt="Figure 1" src="/files/ftp_upload/55748/55748fig1.jpg" /> 
Figure 1: Schematic Diagram of Chamber Assembly. Schematic diagram of chamber assembly: (1) Put the double-layer O-ring-attached coverslip on the stage acceptor. (2) Place the silicon O-ring on the cover slip. (3) Place the base. (4) Lock the sealing locker. (5) Load the sample in the center of the O-ring and place the cover. <a href="http://cloudflare.jove.com/files/ftp_upload/55748/55748fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55748/55748fig2.jpg" /> 
Figure 2: Live Imaging of R3/R4 Differentiation and Rotation. R3/R4 labelled with m&#948;0.5-Gal4 expressing nuclear-form GFP during 10 h of live imaging. All the figures were captured from Movie 1. (A) Image taken at the beginning. (A&#39;-A&#34;) Image enlarged from the boxed area on the left (A&#39;) and right (A&#34;). (B) Image taken at the end of the 10 h period. (B&#39;-B&#34;) Image enlarged from the boxed area on the left (B&#39;) and right (B&#34;). (C-C&#39;&#39;&#39;) A single R3/R4 pair (marked by an arrow in A&#39;) is followed through time to show the rotation of the R3/R4 pair. Scale bars = 15 &#181;m, 10 &#181;m, and 5 &#181;m in A, B, and C, respectively. All the figures are maximum intensity z-projections. The time scale is shown in h:min. <a href="http://cloudflare.jove.com/files/ftp_upload/55748/55748fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/55748/55748mov1.jpg" /> 
Movie 1: <a href="http://cloudflare.jove.com/files/ftp_upload/55748/Movie_1.avi" target="_blank">Please click here to download this movie.</a>
<img alt="Movie 2" src="/files/ftp_upload/55748/55748mov2.jpg" /> 
Movie 1: <a href="http://cloudflare.jove.com/files/ftp_upload/55748/Movie_2.avi" target="_blank">Please click here to download this movie.</a>

Watch this Scientific Journal Video about Long-term Live Imaging of Drosophila Eye Disc at JoVE.com

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.

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 ...

long-term-live-imaging-of-drosophila-eye-disc

Research

JoVE Journal

Developmental Biology

8.4K Views.  National Yang-Ming University.  Dit protocol beschrijft een gedetailleerde methode voor de lange termijn ex vivo cultuur en live beeldvorming van een Drosophila imaginaire disc. Het toont fotoreceptor differentiatie en ommatidial rotatie binnen de 10 uur periode van live-imaging van het oog disc. Het protocol is eenvoudig en niet duur setup nodig. 

Video: Long-term Live Imaging of Drosophila Eye Disc

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 ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Biology

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Extended Live Imaging of Drosophila Pupal Leg Morphogenesis Without Puparium

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Long-term Live Imaging of Drosophila Eye Disc

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