By exploiting the long term imaging potential of this new pupal model together with Drosophila's genetic tractability this method can help us answer key questions in the wound healing and innate immunity fields. The pupal stage offers some distinct advantages for live imaging over more traditional Drosophila wound healing models. For example pupae can imaged over far longer time periods, more tissue area is available for experimental manipulation, and more hemocytes are present at this stage.

In addition the efficiency of RNAi mediated gene inactivation is considerably improved during these pupal stages allowing many genes to be knocked down in a tissue or time specific manner. To set up the pupae collection set up vials with 20 virgin females and 20 males. Then 18 hours before the imaging session collect at least 10 newly-formed white pre-pupae from the vials.

To collect a pupa use forceps or a fine paint brush to dislodge it from the interior vial surface and transfer it carefully to the side of a new fly vial. After the pupae have developed to the desired time point transfer them to a piece of double sided sticky tape mounted on a glass slide. Now under a bright field dissection microscope carefully remove the pupae from their casings.

Position the pupae so that their ventral sides are firmly stuck to the tape. Make the first incision in the anterior-most region of the puparium using the forceps. 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.

Next carefully tear or cut the pupal case open in an anterior to posterior direction using forceps or microscissors until the pupa is completely free from the brown opaque brittle casing. Be careful these pupae are very fragile. Now it is crucial that the forceps and microscissors do not puncture the pupal surface during this step.

Try to avoid touching the pupa itself as you remove the surrounding case. Now mount the undamaged pupae in a glass bottom dish using heptane glue. First deposit a line of prepared heptane glue from a 10 microliter aliquot.

Then after five seconds of drying use forceps to carefully transfer the dissected pupae onto the glue. When learning, line up to five pupae. More can be prepared with gained experience.

Roll the pupae using forceps to mount the pupae so that the wing is flat on the cover glass with the majority of the wing surface in direct contact with the cover glass. Lastly to prevent dehydration add a piece of absorbent filter paper soaked in distilled water to the side of the glass bottom dish. Then cover the dish and proceed with the laser-induced wounding procedure.

Transfer the mounted pupae to a wide field microscope equipped with a tunable laser ablation system. Tune the pulsed-UV air-cooled nitrogen-pumped ablation laser to the desired wavelength using the appropriate dye cell. For optimal wounding set the pulse repetition rate to 40 hertz.

Now using 40 times or 63 times objectives and bright field adjust the microscope stage controls to locate the pupal wing of the first pupa to be wounded. Focus on the plane of the pupal wing epithelium nearest the glass cover slip. Align the area to be wounded with the known target area of the ablation laser.

To do so, take advantage of the crosshair marker within the microscope eyepiece. Next adjust the power of the laser by adjusting the energy density attenuator slide in the microscope. Take advantage of the click stops to make the setting reproducible.

Now to make a wound activate the ablation laser with a quick click of manual control. Then check for the appearance of the transient air bubble at the ablation site which is normal with wounding. If the wounding is unsuccessful try varying the focal plane.

Alternatively gradually increase the laser power using the attenuator slide until the desired wound sizes achieved. After wounding the pupal 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 five to 20%works best to minimize photobleaching. Focus on the whole pupal 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 pupal wing every three 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. The wounded wings of Drosophila pupae at 18 hours after puparium formation were imaged using confocal time lapse microscopy. Laser induced injury to the pupal wing epithelium stimulated a rapid migration of Drosophila innate immune cells called hemocytes to the wound site.

The hemocytes were labeled with both a nuclear marker and a cytoplasmic or cytoskeletal marker to allow tracking and a view of their morphology, respectively. Tracking the hemocyte nuclear trajectories demonstrates the complex spatio-temporal dynamics of the inflammatory response. Within 30 minutes of wounding hemocytes located closest to the injury site have begun directed migration towards the wound.

Eventually hemocytes located progressively further away from the injury begin migrating towards the wound. By utilizing the photo convertible fluorophor Kaede subpopulations of these migratory wing hemocytes were selectively labeled. This method has been used to show that hemocytes recruited to an initial wound are temporarily desensitized to a second wound generated 90 minutes later.

By labeling the E-cadherin within cellular adherens junctions with GFP the wound edge is easily identifiable and this allows analysis of wound healing dynamics. Most wounds begin to re-epithelialize within an hour of injury. The wound edge advances inwards and the wounds ultimately heal within three hours.

Once this technique has been mastered multiple pupae can be prepared for live imaging within just 10 minutes but while attempting this procedure it's important to remember that once dissected from their pupal case Drosophila pupae are extremely vulnerable to damage and dehydration. After its development this technique has paved the way for researchers in the innate immunity field to collaborate with computational biologists and apply more sophisticated mathematical modeling to explore behavior of the wound attractant signals that are responsible for the inflammatory cell recruitment. And even more recently alternative cell types have also been labeled to follow their response to laser-induced injury and this has revealed that adipocyte cells, the Drosophila fat-body cells, also respond to wounds within this system.

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