Encyclopedia of Experiments: Immunology
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Transcript
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.
