Team:Hannover/Protocols/Detection/Confocal

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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols" target="_blank">Protocols</a> / Confocal Microscopy</h1>
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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols">Protocols</a> / Confocal Microscopy</h1>
<table colspan="2"><tr><td><h4>Material:</h4></td>
<table colspan="2"><tr><td><h4>Material:</h4></td>
<tr><td>Prepared MS Samples</td><td></td></tr>
<tr><td>Prepared MS Samples</td><td></td></tr>
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<tr><td>Confocal Microscope</td><td>(here:Eclipse TE2000-E, Nikon, Japan)</td></tr>
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<tr><td>Confocal Microscope</td><td>(here: Eclipse TE2000-E, Nikon, Japan)</td></tr>
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<tr><td></td><td>Settings</u></b><br><p><ol><li>100 x amplification<li>For excitation of fluorescence for confocal pictures using lasers with λ= 408 nm and λ= 488 nm <br><li>Transmission microscopy: halogen lamp<li>Detection of GFP signals: mercury lamp</li></ol></td></tr></table>
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<tr><td></td><td><b><u>Settings</u></b><br><p><ol><li>100 x magnification<li>For penetration of fluorescence the laser wavelength were adjusted to λ= 408 nm and λ= 488 nm <br><li>Transmission microscopy: halogen lamp <u>vs.</u> Detection of GFP signals: mercury lamp</li></ol></td></tr></table>
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<br><br>
<h2>Protocol</h2>
<h2>Protocol</h2>
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<p class="text">Before samples are screened by powerful lasers, which could bleach the GFP signal, the mercury based transmission light might be used for browsing the sample. After the right sample layer was focused, the source of lightening was switched to the lasers and the pinhole value needed to be adjusted. Three pictures were taken: one in the green channel, one in the red channel and one of in pseudo-transmission light. GFP molecules gave a green signal, while the chlorophyll visualized the chloroplasts as red dots. In order to compare the pictures, a scale bar was added in the pictures.</p>   
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<p class="text">Before samples are screened by powerful lasers, which could bleach the GFP signal, the halogen transmission light was used for browsing the sample. After the right sample layer was focused, the source of lightening was switched to the lasers (mercury) and the pinhole-value needed to be adjusted. Three pictures were taken: one in the green channel, one in the red channel and one of in pseudo-transmission light. GFP molecules gave a green signal, while the chlorophyll visualized the chloroplasts as red dots. In order to compare the pictures, a scale bar was added in the pictures.</p>   
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Latest revision as of 23:38, 17 October 2014

Protocols / Confocal Microscopy

Material:
Prepared MS Samples
Confocal Microscope(here: Eclipse TE2000-E, Nikon, Japan)
Settings

  1. 100 x magnification
  2. For penetration of fluorescence the laser wavelength were adjusted to λ= 408 nm and λ= 488 nm
  3. Transmission microscopy: halogen lamp vs. Detection of GFP signals: mercury lamp


Protocol

Before samples are screened by powerful lasers, which could bleach the GFP signal, the halogen transmission light was used for browsing the sample. After the right sample layer was focused, the source of lightening was switched to the lasers (mercury) and the pinhole-value needed to be adjusted. Three pictures were taken: one in the green channel, one in the red channel and one of in pseudo-transmission light. GFP molecules gave a green signal, while the chlorophyll visualized the chloroplasts as red dots. In order to compare the pictures, a scale bar was added in the pictures.






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