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UV laser mediated cell selective destruction by confocal microscopy.

Soustelle L, Aigouy B, Asensio ML, Giangrande A - Neural Dev (2008)

Bottom Line: Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy.We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers.Coupling this technique with time-lapse enables the destruction and following of any cell(s) in any pattern(s) in living animals as well as in cell culture systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique et Biologie Moléculaire et Cellulaire, IGBMC/CNRS/ULP/INSERM - BP 10142 67404 ILLKIRCH, c.u. de Strasbourg, France. lsoustel@titus.u-strasbg.fr

ABSTRACT
Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy. We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers. Coupling this technique with time-lapse enables the destruction and following of any cell(s) in any pattern(s) in living animals as well as in cell culture systems.

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High resolution images of confocal assisted UV laser selective destruction. (a,b) Confocal images of repo::GFP expressing cells prior to (a) and after (b) selective destruction. (b) The proximal glial cell (p) has been targeted for selective destruction; the distal cell (d) was not targeted. (c,d) actin-GFP labeling in the fly wing epithelium prior to (c) and just after (d) UV irradiation. The region of interest defined by the 'Point bleach' function is indicated by the black square in (c). (d) Notice that, upon irradiation, GFP labeling is specifically absent in the targeted nucleus. Images to the bottom of (c,d) correspond to Z optical sections taken along the axis indicated by the black arrowheads. Color coding is used to quantify GFP labeling: blue (0) corresponds to background, red (250) to high levels. (e-h) actin-GFP labeling in the fly wing epithelium prior to (e) and 10 (f), 22 (g) or 32 minutes (h) after UV irradiation. The two targeted cells (indicated by red arrows in (e)) are absent 10 minutes after UV-mediated destruction (indicated by white asterisks in (f)). The space previously occupied by the targeted cells is subsequently occupied by the neighboring cells (g,h). See also Additional file 5. Scale bars: (a,b) 25 μm; (c,d) 5 μm; (e-h) 10 μm.
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Figure 1: High resolution images of confocal assisted UV laser selective destruction. (a,b) Confocal images of repo::GFP expressing cells prior to (a) and after (b) selective destruction. (b) The proximal glial cell (p) has been targeted for selective destruction; the distal cell (d) was not targeted. (c,d) actin-GFP labeling in the fly wing epithelium prior to (c) and just after (d) UV irradiation. The region of interest defined by the 'Point bleach' function is indicated by the black square in (c). (d) Notice that, upon irradiation, GFP labeling is specifically absent in the targeted nucleus. Images to the bottom of (c,d) correspond to Z optical sections taken along the axis indicated by the black arrowheads. Color coding is used to quantify GFP labeling: blue (0) corresponds to background, red (250) to high levels. (e-h) actin-GFP labeling in the fly wing epithelium prior to (e) and 10 (f), 22 (g) or 32 minutes (h) after UV irradiation. The two targeted cells (indicated by red arrows in (e)) are absent 10 minutes after UV-mediated destruction (indicated by white asterisks in (f)). The space previously occupied by the targeted cells is subsequently occupied by the neighboring cells (g,h). See also Additional file 5. Scale bars: (a,b) 25 μm; (c,d) 5 μm; (e-h) 10 μm.

Mentions: After 20 seconds of UV irradiation (power at maximum values, speed 400 Hz), GFP labeling rapidly fades (within minutes) and never resumes, even after several hours (Figure 1a,b), suggesting that the targeted cell has died. Death induced by 20 seconds of UV irradiation has been confirmed by lack of glial-specific labeling in the targeted cell (Additional file 2; see also [16]). Altogether, these data strongly suggest that UV laser targeting leads to cell destruction. To formally conclude that cell death occurs only in the irradiated cell, we have performed several control experiments.


UV laser mediated cell selective destruction by confocal microscopy.

Soustelle L, Aigouy B, Asensio ML, Giangrande A - Neural Dev (2008)

High resolution images of confocal assisted UV laser selective destruction. (a,b) Confocal images of repo::GFP expressing cells prior to (a) and after (b) selective destruction. (b) The proximal glial cell (p) has been targeted for selective destruction; the distal cell (d) was not targeted. (c,d) actin-GFP labeling in the fly wing epithelium prior to (c) and just after (d) UV irradiation. The region of interest defined by the 'Point bleach' function is indicated by the black square in (c). (d) Notice that, upon irradiation, GFP labeling is specifically absent in the targeted nucleus. Images to the bottom of (c,d) correspond to Z optical sections taken along the axis indicated by the black arrowheads. Color coding is used to quantify GFP labeling: blue (0) corresponds to background, red (250) to high levels. (e-h) actin-GFP labeling in the fly wing epithelium prior to (e) and 10 (f), 22 (g) or 32 minutes (h) after UV irradiation. The two targeted cells (indicated by red arrows in (e)) are absent 10 minutes after UV-mediated destruction (indicated by white asterisks in (f)). The space previously occupied by the targeted cells is subsequently occupied by the neighboring cells (g,h). See also Additional file 5. Scale bars: (a,b) 25 μm; (c,d) 5 μm; (e-h) 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2387153&req=5

Figure 1: High resolution images of confocal assisted UV laser selective destruction. (a,b) Confocal images of repo::GFP expressing cells prior to (a) and after (b) selective destruction. (b) The proximal glial cell (p) has been targeted for selective destruction; the distal cell (d) was not targeted. (c,d) actin-GFP labeling in the fly wing epithelium prior to (c) and just after (d) UV irradiation. The region of interest defined by the 'Point bleach' function is indicated by the black square in (c). (d) Notice that, upon irradiation, GFP labeling is specifically absent in the targeted nucleus. Images to the bottom of (c,d) correspond to Z optical sections taken along the axis indicated by the black arrowheads. Color coding is used to quantify GFP labeling: blue (0) corresponds to background, red (250) to high levels. (e-h) actin-GFP labeling in the fly wing epithelium prior to (e) and 10 (f), 22 (g) or 32 minutes (h) after UV irradiation. The two targeted cells (indicated by red arrows in (e)) are absent 10 minutes after UV-mediated destruction (indicated by white asterisks in (f)). The space previously occupied by the targeted cells is subsequently occupied by the neighboring cells (g,h). See also Additional file 5. Scale bars: (a,b) 25 μm; (c,d) 5 μm; (e-h) 10 μm.
Mentions: After 20 seconds of UV irradiation (power at maximum values, speed 400 Hz), GFP labeling rapidly fades (within minutes) and never resumes, even after several hours (Figure 1a,b), suggesting that the targeted cell has died. Death induced by 20 seconds of UV irradiation has been confirmed by lack of glial-specific labeling in the targeted cell (Additional file 2; see also [16]). Altogether, these data strongly suggest that UV laser targeting leads to cell destruction. To formally conclude that cell death occurs only in the irradiated cell, we have performed several control experiments.

Bottom Line: Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy.We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers.Coupling this technique with time-lapse enables the destruction and following of any cell(s) in any pattern(s) in living animals as well as in cell culture systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique et Biologie Moléculaire et Cellulaire, IGBMC/CNRS/ULP/INSERM - BP 10142 67404 ILLKIRCH, c.u. de Strasbourg, France. lsoustel@titus.u-strasbg.fr

ABSTRACT
Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy. We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers. Coupling this technique with time-lapse enables the destruction and following of any cell(s) in any pattern(s) in living animals as well as in cell culture systems.

Show MeSH