the photoconversion technique for exploring inflammatory cell dynamics in insect pupae

The Photoconversion Technique for Exploring Inflammatory Cell Dynamics in Insect Pupae

After wounding the pupil wing margins, quickly transfer the glass-bottomed dish to an appropriate microscope for time-lapse imaging. Then, open the appropriate image capture software.
In the software, turn on the appropriate lasers, and adjust their power and gain offset to get enough fluorescent signal without pixel saturation. Generally, the lowest possible laser power in the range 5% to 20% works best to minimize photobleaching.
Focus on the whole pupil wing under low magnification or focus on the wound under high magnification to investigate wound repair. To capture both the repairing epithelium and inflammatory cell recruitment, first, set the microscope to record a z-stack using the fine focus adjustment on the control panel. Scan from the wounded epithelium through to the extracellular space beneath, containing migrating hemocytes.
Next, set the software to record z-slices through the pupil wing every 3 microns or at even tighter intervals. For time-lapse imaging, record z-stacks at least every 30 seconds for at least one hour.
When using the photo-convertible probes to selectively photo-convert and label a subset of cells during imaging, open the appropriate modules within the imaging software to perform the photo-conversion and activate the 405-nanometer laser. Then, select the cells to be photo-converted within the FRAP software using a selection tool.
Next, set the time course for photo-conversion to a single iteration frame, and set the 405-nanometer laser to 20% laser power, then click Start The Experiment to perform photo conversion. Once completed, exit the FRAP module and return to the original imaging screen. There, tune the lasers to the fluorophores in use and image the photo-converted and non-photo-converted cells using time-lapse recordings.

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This protocol consists of four main sequential steps: (1) Preparation of&#160;Drosophila&#160;stocks and staging of&#160;Drosophila&#160;pupae, (2) Pupal dissection and mounting, (3) Pupal wounding, (4)&#160;in vivo&#160;time-lapse confocal imaging.
1. Preparation of Drosophila Stocks and Staging of Pupae
<ol>
	<li>Obtain appropriate&#160;Drosophila&#160;stocks (see Introduction and&#160;Table of Materials).</li>
	<li>Collect young healthy adult flies of the appropriate genotype.
	<ol>
		<li>Select adult flies by using carbon dioxide gas pads to briefly anesthetize the flies and a fine paintbrush to transfer flies of the appropriate genotype or gender to a collection vial.</li>
		<li>Add 20 virgin females and 20 males to each vial containing standard fly food media (a cornmeal-molasses-agar mixture, see&#160;Table of Materials) supplemented with yeast.</li>
	</ol>
	</li>
	<li>For optimal pupal generation, tip the adult flies each day onto new food in fresh vials, keeping all vials at 25 &#176;C. 
	NOTE: If the Gal4-upstream activating sequence (Gal4-UAS) system is being used to drive lineage-specific gene expression, all steps should be performed at 25 &#176;C or above, as the Gal4-UAS system is temperature sensitive.</li>
	<li>18 h before the scheduled imaging session, select at least 10 newly formed white pre-pupae (Figure 1A) from the vials (i.e.&#160;0 h after puparium formation, APF) using forceps or a fine paintbrush to dislodge pupae from the interior vial surface and transfer pupae carefully to the side of a clean empty plastic vial. 
	NOTE: Wandering 3rd&#160;instar larvae crawl upwards out of the food medium to undergo pupation; newly formed white prepupae are easily identified as they possess everted anterior spiracles and are stationary (unlike 3rd&#160;instar larvae). The cuticle transforms into the &#39;puparium&#39; (the pupal case) which is initially soft and white. Care must be taken to avoid damaging the pupae, as this can lead not only to an unwanted injury-induced inflammatory response but also to a significant developmental delay.</li>
	<li>Age the selected pupae to the appropriate developmental stage (18 h APF will give optimal results) in the vial at 25 &#176;C. 
	NOTE: As the pupae develop, the pupal case will become progressively darker and more brittle.</li>
	<li>Prepare other reagents for the next step ahead of time. To make the heptane glue, combine a 20 cm length of rolled-up double-sided tape with 20 mL heptane in a 50 mL centrifuge tube, seal with the paraffin film, and rock at room temperature overnight on the bench.</li>
</ol>
2. Preparation and Dissection of&#160;Drosophila&#160;Pupae
<ol>
	<li>Transfer the staged&#160;Drosophila&#160;pupae to a piece of double-sided sticky tape mounted on a glass slide. Position the pupae so that the ventral side is firmly stuck to the tape and the dorsal side is facing upwards (Figure 1B). 
	NOTE: The anterior of the pupa will be identifiable from the two spiracles protruding from the anterior end of the pupal case.</li>
	<li>Carefully remove pupae from their protective puparium casing under a brightfield dissection microscope using forceps and micro-scissors (Figure 1B-D).
	<ol>
		<li>Initially, make an incision in the anterior-most region of the puparium using the forceps (Figure 1B). Ensure that the pupal case in this area is hollow and devoid of pupal tissue because the pupae will have shrunk within the case during early pupal development.</li>
		<li>After this initial incision, carefully tear or cut the pupal case open in an anterior-to-posterior direction using forceps or microscissors (Figure 1C) until the pupa is completely free from the brown opaque brittle casing (Figure 1D). 
		NOTE: Pupae at this stage are very fragile and care must be taken to avoid puncturing the pupal surface; puncturing is obvious as hemolymph quickly leaks out from the puncture site. Pupae with punctures, however small, should be discarded.</li>
	</ol>
	</li>
	<li>Mount the pupae in a glass-bottomed dish using heptane glue.
	<ol>
		<li>Use a 20 mL pipette tip to place a 10 mL drop of pre-prepared heptane glue (see section 1.6 above) in a line on the glass-bottomed dish.</li>
		<li>Allow the glue to dry for 5 s before transferring the dissected pupae carefully onto the heptane glue using forceps (Figure 1E).</li>
		<li>For ease of wounding and imaging, line the pupae up in a row; approximately 5 pupae are suggested but more will be manageable with experience. 
		NOTE: The use of heptane glue is recommended when using upright imaging systems but is not necessary when using an inverted system. If the glue creates optical aberrations during imaging, pupae can be placed directly on the coverglass instead &#8211; the natural adhesion between the pupal tissue and glass will be sufficient in most cases for stable imaging, as long as care is taken when moving the imaging dish between microscopes.</li>
	</ol>
	</li>
	<li>For best results, mount the pupae so that the wing is flat on the coverglass with the majority of the wing surface in direct contact with the coverglass (Figure 1F). Roll the pupae using forceps to change their position to ensure the wing is mounted correctly.</li>
	<li>To prevent sample dehydration during the imaging period, add a piece of absorbent filter paper soaked in distilled water to the side of the glass-bottomed dish at the end of mounting, being careful not to disturb the pupae (Figure 1E). Cover the dish with a lid.&#160;&#160; 
	NOTE: Pupae are now ready for wounding and imaging.</li>
</ol>
3. Laser-induced Wounding of&#160;Drosophila&#160;Pupal Wings
<ol>
	<li>Transfer the glass-bottomed dish containing the mounted pupae to a wide-field microscope equipped with a tunable laser ablation system.
	<ol>
		<li>Use a pulsed-UV air-cooled nitrogen-pumped ablation laser tuned to 435 nm &#8211; see&#160;Table of Materials&#160;for details; the precise wavelength of the light used for illumination is selected by the user via an appropriate dye cell.</li>
	</ol>
	</li>
	<li>Using brightfield optics, adjust the microscope stage controls to locate the pupal wing of the first pupa to be wounded (Figure 1F).</li>
	<li>For optimal results, use an oil immersion 40X or 63X objective lens for both the imaging and laser ablation; ensure that the immersion liquid used (oil or glycerol) is consistent between ablation and imaging systems. Adjust the microscope to focus on the plane of the pupal wing epithelium nearest the glass coverslip (i.e.&#160;focus on the region of the epithelium to be wounded) using the fine focus control knob.</li>
	<li>Using the microscope stage adjustments, position the pupal wing so that the area to be wounded is directly aligned with the known target area of the ablation laser.</li>
	<li>Use an energy density attenuator slide fitted to the microscope to manually adjust the power level of the ablation light. 
	NOTE: The attenuator slider has click stops and rulings to identify the relative level of attenuation and permit the use of reproducible settings.</li>
	<li>Using an external manual&#160;trigger&#160;control, activate the ablation laser using a single brief click of the trigger to make a wound. Check for the appearance of the transient air bubble at the ablation site since wounding will normally be accompanied by it. Check if the laser-induced wounding has been successful using the appropriate fluorescent filters to visualize the pupal epithelium.&#160;&#160;&#160; 
	NOTE: Take care to avoid accidental exposure to the laser beams as the beam reflections can cause severe eye or skin damage.</li>
	<li>If wounding is unsuccessful, vary the focal plane (moving the microscope focus slightly above or below the current focal level) and repeat the single click of the ablation trigger. Alternatively, gradually increase the laser power using the attenuator slide until the desired wound size is achieved.</li>
	<li>To vary the ablation laser pulse repetition rate, use the&#160;Repetition rate&#160;knob on the rear control panel (which changes the rate from less than 1 pulse/s up to 60 Hz). For optimal wounding, set the pulse repetition rate to 40 Hz.</li>
	<li>To generate different-sized wounds, use the energy density attenuator slide to manually adjust the power level of the ablation light.</li>
	<li>Refrain from wounding all of the mounted pupae and use these non-ablated pupae as unwounded controls.</li>
	<li>For consistent results, regularly realign the ablation system (using the relevant operating manual). Also, clean and refill the dye resonator cell that controls the laser output wavelength.</li>
</ol>
4. In Vivo&#160;Time-lapse Confocal Imaging
<ol>
	<li>Quickly transfer the glass-bottomed dish to an appropriate microscope for time-lapse imaging.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: For optimal results, use a high-specification confocal or spinning disc microscope equipped with sensitive detectors that can detect both green fluorescent protein (GFP) and mCherry fluorophores.</li>
	<li>To image the entire pupal wing, use a low magnification (e.g.&#160;20X) objective lens (Figure 2A). To image wound repair and the accompanying inflammatory response with high spatial resolution, use the oil immersion 40X (NA 1.3) or 63X (NA 1.4) objective lenses (see representative images in&#160;Figure 2B-D).</li>
	<li>Open the appropriate image capture software associated with the microscope.</li>
	<li>Using the image capture software, turn on the appropriate lasers&#160;e.g.&#160;the 488 nm and 561 nm lasers to visualize GFP and mCherry fluorophores, respectively (by clicking in the relevant boxes) and adjust the laser power and gain/offset settings to give sufficient fluorescent signal whilst avoiding pixel saturation; use the lowest possible laser power (in the range 5 - 20%) to minimize photobleaching and phototoxicity.</li>
	<li>To capture both the repairing epithelium and inflammatory cell recruitment, set the microscope to record a z-stack using the fine focus adjustment knobs on the control panel; for optimal results, set the software (using manual button-clicks) to record z-slices through the pupal wing (minimum every 3 mm), from the top of the wounded epithelium through to the extracellular space beneath (containing migrating hemocytes) to achieve a large z-stack (in the range of 50 -&#160;100 mm).</li>
	<li>For time-lapse imaging, record z-stacks at regular time intervals (minimum every 30 s) for at least 1 h post-wounding.&#160; 
	NOTE: The exact time interval between the z-stacks chosen represents a trade-off between capturing the rapidly changing cell dynamics and avoiding photo-bleaching of the samples.</li>
	<li>To simultaneously image multiple pupae (including non-ablated unwounded controls), use a motorized stage (attached to the microscope) and the multi-position acquisition feature available within the imaging software. Manually set the position of each pupa within the software using the stage position control knobs and then manually set the appropriate z-stack limits (top and bottom) for each pupa.</li>
	<li>Visualize the time-lapse images either during image capture or later with specialist image analysis software (such as ImageJ) using z-stack projections or 3-D rendering. For example, to follow the movements of individual hemocytes (as in&#160;Figure 2C&#39;&#160;and&#160;D&#39;), track hemocyte nuclei using the open-access ImageJ plug-ins &#34;TrackMate&#34; or &#34;Manual Tracking&#34; (methods published in).</li>
	<li>Use photoconvertible probes (such as Kaede) to selectively photoconvert and label a subset of epithelial or immune cells during imaging.
	<ol>
		<li>Open appropriate modules within the imaging software to perform the photoconversion (such as the Fluorescence recovery after photobleaching (FRAP), and fluorescence recovery after photo-bleaching module) and activate the 405nm laser (by clicking in the relevant software box).</li>
		<li>Select cells to be photoconverted within the FRAP software using the square, circular, or freehand selection tool. Within the FRAP software, set the time course for photoconversion (Bleaching) to a single iteration/frame and set the 405 nm laser at 20% laser power. Manually click&#160;Start the experiment&#160;to perform photoconversion.</li>
		<li>Exit the FRAP module (click&#160;Close) and return to the original imaging screen within the software; use the 488 nm and 561 nm lasers to image the behavior of photoconverted and non-photoconverted cells by setting up a z-stack and time-lapse recording as above. 
		NOTE: Photoconverted probes remain stable for many hours after the initial photoconversion, enabling the behavior of the photoconverted cells to be followed over time (for at least 5 h). For example, inflammatory cells in the wound can be selectively photoconverted (Figure 2F) and their behavior followed as they resolve from the injury site (Figure 2G&#160;and H).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Drosophila&#160;stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ubiquitous GFP-tagged E-cadherin;Ubi-p63E-shg.GFP; (chrII)</td>
			<td>Kyoto Stock Center, DGRC</td>
			<td>#109007</td>
			<td>Ubi-p63E promoter sequences drive the expression of&#160;Drosophila&#160;E-cadherin (shotgun) tagged at the C-terminal end with GFP.</td>
		</tr>
		<tr>
			<td>Ubiquitous GFP-tagged E-cadherin;;Ubi-p63E-shg.GFP (III)</td>
			<td>Bloomington&#160;Drosophila&#160;Stock Centre (Indiana University)</td>
			<td>#58742</td>
			<td>Ubi-p63E promoter sequences drive the expression of&#160;Drosophila&#160;E-cadherin (shotgun) tagged at the C-terminal end with GFP.</td>
		</tr>
		<tr>
			<td>Ubiquitous GFP-tagged Moesin P{sGMCA}3.1</td>
			<td>Bloomington&#160;Drosophila&#160;Stock Centre (Indiana University)</td>
			<td>#59023</td>
			<td>The ubiquitously expressed sqh promoter/enhancer drives expression of a fragment of Moesin (that includes the actin binding sequences) tagged with GFPS65T.</td>
		</tr>
		<tr>
			<td>Hemocyte specific serpent-Gal4 driver ;srp-Gal4;</td>
			<td>Generated by Katja Bruckner</td>
			<td>Generated by Katja Bruckner</td>
			<td>The expression of Scer\GAL4 fused to a polyA tail is controlled by 2 genomic sequences from upstream of&#160;Drosophila&#160;serpent. Ref: Br&#252;ckner, K., Kockel, L., Duchek, P., Luque, C.M., R&#248;rth, P., Perrimon, N. The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell. 7 (1), 73&#8211;84, doi: 10.1016/j.devcel.2004.06.007 (2004).</td>
		</tr>
		<tr>
			<td>UAS-nuclearRFP&#160; w1118;;P{UAS-RedStinger}6</td>
			<td>Bloomington&#160;Drosophila&#160;Stock Centre (Indiana University)</td>
			<td>#8545 or #8547</td>
			<td>UAS regulatory sequences drive expression of the DsRed.T4 form of RFP which is tagged at the C-terminal end with a nuclear localisation signal</td>
		</tr>
		<tr>
			<td>UAS-cytoplasmicGFP ;;P{UAS-GFP.S65T}</td>
			<td>Bloomington&#160;Drosophila&#160;Stock Centre (Indiana University)</td>
			<td>Multiple stocks available (e.g. #1522)</td>
			<td>Expression of the S65T version of GFP by UAS regulatory sequences; the S65T variant exhibits increased brightness.</td>
		</tr>
		<tr>
			<td>UAS-photoconvertibleKaede&#160; w1118;; P{UAS-Kaede.A}3</td>
			<td>Bloomington&#160;Drosophila&#160;Stock Centre (Indiana University)</td>
			<td>#26161</td>
			<td>Kaede protein emits bright green fluorescence after synthesis, but changes efficiently to a bright stable red fluorescence on irradiation with UV.</td>
		</tr>
		<tr>
			<td>GFP-tagged spaghetti squash&#160; w1118;;P{sqh-GFP.RLC}</td>
			<td>Bloomington&#160;Drosophila&#160;Stock Centre (Indiana University)</td>
			<td>#57145</td>
			<td>The sqh coding region, which is tagged at the C-terminal end with a T:Avic\GFPS65T&#160;tag, is expressed under the control of the natural sqh promoter.</td>
		</tr>
		<tr>
			<td>Ingredients for fly food media</td>
			<td></td>
			<td></td>
			<td>Fly food media is made according to standard procedures (see Greenspan, R. 1997. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Press. 1-191 pp.)</td>
		</tr>
		<tr>
			<td>Maize</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>Soya flour</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>Malt extract</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>Molasses</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>Difco agar</td>
			<td>BD Biosciences, Fisher Scientific</td>
			<td>DF0142-15-2</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Sigma</td>
			<td>402907</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Nipagen</td>
			<td>Sigma</td>
			<td>79721</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Dried baker&#39;s yeast</td>
			<td>Redstar, Dutscher Scientific, UK LTD</td>
			<td>Redstar, Dutscher Scientific, UK LTD</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Sample preparation and mounting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Fine sable paintbrush</td>
			<td>Daler-Rowney (or equivalent)</td>
			<td>#0 or 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific (or Fine Science Tools)</td>
			<td>NC9404145</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Glass bottomed dishes for imaging</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td>We suggest using 35mm petri dishes, with at least a 10mm Microwell, 0.085-0.13mm cover glass, uncoated. Dishes with larger microwells will enable increasing numbers of pupae to be mounted and imaged in a single experiment.</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma</td>
			<td>51730-5ML</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Double sided sticky tape (e.g. Scotch)</td>
			<td>Agar Scientific</td>
			<td>AGG263</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>50ml tube (for heptane glue)</td>
			<td>Falcon tubes from Fisher Scientific</td>
			<td>14-432-22</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Agar Scientific</td>
			<td>AGL4244</td>
			<td>For dissection of&#160;Drosophila&#160;pupae</td>
		</tr>
		<tr>
			<td>Dissecting stereo microscope with brightfield</td>
			<td>Leica (or equivalent)</td>
			<td>M50</td>
			<td>For dissection of&#160;Drosophila&#160;pupae</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>John Weiss International</td>
			<td>103123</td>
			<td>Miniature Research Scissors (straight)</td>
		</tr>
		<tr>
			<td>Laser ablation and imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitogen ablation laser</td>
			<td>Spectra-Physics (or Andor equivalent)</td>
			<td>Model VSL-337ND-S</td>
			<td>For wounding, this should be attached to a widefield imaging system</td>
		</tr>
		<tr>
			<td>Multilaser confocal laser-scanning microscope (CLSM)</td>
			<td>Leica (or equivalent)</td>
			<td>TCS AOBS SP8 or SP5-II attached to a Leica DMi8 inverted epifluorescence microscope (or equivalent)</td>
			<td>Ideally including a motorised stage for multi-site and &#39;mosaic&#39; scanning, plus &#39;hybrid&#39; GaAsP detectors (that offer much greater sensitivity and boosting of low signal)</td>
		</tr>
		<tr>
			<td>Environmental chamber</td>
			<td>Life Imaging Services (or equivalent)</td>
			<td>&#34;Microscope Temperature Control System&#34;</td>
			<td>Attached to Confocal microscope for temperature control during imaging</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FRAP software module</td>
			<td>Leica (or equivalent)</td>
			<td>CLSM FRAP software module</td>
			<td>For performing photoconversion of photoconvertible fluorophores such as Kaede</td>
		</tr>
		<tr>
			<td>ImageJ (image analysis software)</td>
			<td>National Institutes of Health (NIH)</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td>Schneider, C.A., Rasband, W.S., Eliceiri, K.W. &#34;NIH Image to ImageJ: 25 years of image analysis&#34;. Nature Methods 9, 671-675, 2012.</td>
		</tr>
		<tr>
			<td>ImageJ plugin &#34;Manual Tracking&#34;</td>
			<td>National Institutes of Health (NIH)</td>
			<td>https://imagej.net/Manual_Tracking</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ plugin &#34;TrackMate&#34;</td>
			<td>ImageJ, NIH</td>
			<td>https://imagej.net/TrackMate</td>
			<td>Tinevez, JY.; Perry, N. &#38; Schindelin, J. et al. (2016), &#34;TrackMate: An open and extensible platform for single-particle tracking.&#34;, Methods 115: 80-90, PMID 27713081</td>
		</tr>
		<tr>
			<td>Volocity (high performance 3D imaging software)</td>
			<td>Perkin Elmer</td>
			<td>Volocity 6.3</td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>IMARIS (image analysis software)</td>
			<td>Bitplane</td>
			<td>IMARIS for Cell Biologists</td>
			<td>For image analysis</td>
		</tr>
	</tbody>
</table>

Source: Weavers, H. et al., <a href="https://app.jove.com/v/57871">Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae.</a>&#160;J. Vis. Exp. (2018)
The video demonstrates the use of photoconversion to study inflammatory cell dynamics in Drosophila pupae. Green hemocytes are drawn to the wounded epithelium, followed by distant migration. Photoconversion shifts certain hemocytes from green to red, facilitating their movement tracking.

<img alt="Figure 1" src="/files/ftp_upload/21878/21878_Figure1.jpg" /> 
Figure 1:&#160;Drosophila pupa preparation for wounding and live-imaging.&#160;(A)&#160;Drosophila&#160;white prepupae collected at 0 h APF, with the anterior end indicated by everted breathing appendages (spiracles). (B) After raising white 0h APF prepupae for 18 h at 25 &#176;C, the puparium appears brown. Dissection of the pupal case should b...

This protocol consists of four main sequential steps: (1) Preparation of&#160;Drosophila&#160;stocks and staging of&#160;Drosophila&#160;pupae, (2) Pupal ...

Begin with a glass-bottomed dish containing transgenic Drosophila pupae carrying labeled wounded epithelium with their cell surface proteins exhibiting green fluorescence.
The pupae&#39;s immune cells &#8212; or hemocytes express photoconvertible green fluorophores in the cytoplasm and red fluorescent proteins in the nucleus, facilitating cell tracking.
The wounded cells release chemoattractants &#8212; or inflammatory molecules, attracting the hemocytes to the wounded site.
Confocal microscopy reveals the wounded area encircled by green epithelial cells.
During healing, green hemocytes with red nuclei near the wound extend membrane protrusions &#8212; filopodia and lamellipodia and migrate toward the wounded area.
Over time, as the chemoattractant diffuses, distant hemocytes migrate toward the wound, creating an immune cell wave.
Illuminate the subpopulation of migratory hemocytes with a 405-nanometer wavelength. This irreversibly converts the hemocytes&#39; photoconvertible fluorophore from green to red.&#160;
These photoconverted hemocytes repair the wounded epithelium and move away from the wound site, differentiating the photoconverted hemocytes from non-photoconverted hemocytes, and elucidating inflammatory cell dynamics.

16 次观看. 探索昆虫蛹炎症细胞动力学的光转化技术

视频: 探索昆虫蛹炎症细胞动力学的光转化技术 - 实验

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

科研

JoVE Journal

发育生物学

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

The Photoconversion Technique for Exploring Inflammatory Cell Dynamics in Insect Pupae

成績單

The Photoconversion Technique for Exploring Inflammatory Cell Dynamics in Insect Pupae