Team:Hannover/Protocols/Detection/Confocal

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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols">Protocols</a> / Confocal Microscopy</h1>
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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols" target="_blank">Protocols</a> / How To: Confocal Microscopy</h1>
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<p><h4>Protocol:</h4></p>
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<table colspan="2"><tr><td><h4>Material:</h4></td>
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<p class="text">After induction of heterologous T4MBP expression, bacterial cell cultures were centrifuged for 15 min at 4500 x g and 4 °C. While the supernatant was decanted, the precipitated bacteria were dissolved in 20 ml isosmotic buffer. According to the harm of heavy metals, all medium supernatants were stored and had to be picked up by professional disposal service. Starting with the dissolving the precipitate, the washing is repeated four more times. Due to the heavy metals, the supernatants obtained here had to be stored also. As the last step, precipitated bacteria were dissolved in 1 ml H20 and dried for 2 d at 60 °C. For latter MS, the reaction tube´s dry-weight before and after loading the aqueous bacteria solution had to be determined.</p>
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<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></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>
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<h2>Protocol</h2>
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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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