Methodology
UV laser mediated cell selective destruction by confocal microscopy
Institut de Génétique et Biologie Moléculaire et Cellulaire, IGBMC/CNRS/ULP/INSERM – BP 10142 67404 ILLKIRCH, c.u. de Strasbourg, France
Neural Development 2008, 3:11 doi:10.1186/1749-8104-3-11
Published: 28 April 2008Additional files
Additional file 1:
Laser power measurement. (a-e) To measure the laser power, we placed the detector head of a power meter (a) in a special 'slide-shaped' device (b), as shown in (c). The special head detector-containing slide-shaped device is then placed on the confocal stage (d) to measure the laser power with a NovaII power meter (Ophir) (arrow in (e)). With this setup, the head detector is illuminated like a classic slide for UV cell destruction experiments. To determine a reproducible position for the measurement, we placed the Leica 63X HCX Plan Apo CS, NA 1.4, lambda blue objective at its upper position. Power measurements after complete recalibration of the whole system (confocal microscope, UV laser and power meter) are 186 μW at 351 nm and 168 μW at 364 nm. A more reproducible and universal measurement (objective independent, see Materials and methods) at the back aperture of the objective gives a value of 2.3 mW.
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Additional file 2:
Confocal assisted UV laser selective cell destruction. (a,b) Confocal images of repo::GFP expressing cells prior to (a) and after (b) selective destruction of glial cells in a developing Drosophila wing (indicated by the dotted area in (a,b)). Distal is to the right. (a) Cells targeted for selective destruction are indicated by arrows. (b) After UV irradiation (17 hours after puparium formation), GFP labeling rapidly faded, suggesting that targeted cells die (indicated by asterisks). (c) Repo immunolabeling on the same dissected wing at 22 hours after puparium formation. Cell death is confirmed by lack of Repo glial-specific labeling in the targeted cells (indicated by asterisks) whereas other glial cells (included in the dotted area) were not affected.
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Wing glia GFP bleaching. Time-lapse sequence showing that strong bleaching conditions (using a 488 nm beam) do not produce any apparent damage as targeted cells divide and migrate just as non-targeted cells, therefore behaving as wild-type cells.
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Wing glia mild UV irradiation. Time-lapse sequence showing that by using mild UV irradiation, the targeted cell, which retains some GFP expression, undergoes rounding, fragmentation and loses contact with neighbor cells.
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Epithelial rearrangement after cell destruction. Time-lapse sequence showing that only the targeted cell lacks GFP labeling, and is destroyed upon UV irradiation. Note that the space occupied by the targeted cells is eventually occupied by its neighbors.
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Selective destruction along the Z axis. Time-lapse sequence showing that GFP-positive cells located on the dorsal epithelial sheet are not affected by UV-mediated cell destruction of ventrally located cells.
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Confocal assisted cell destruction of HeLa cells. Time-lapse sequence showing that UV-mediated destruction of HeLa cells induces apoptosis, as confirmed by S-FLICA labeling.
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