Team:Heidelberg/Notebook measure/NotebookMi
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__NOTOC__ | __NOTOC__ | ||
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{{Template_HD_3}} | {{Template_HD_3}} | ||
<html><body id="Notebook_natprom"></body></html> | <html><body id="Notebook_natprom"></body></html> | ||
{| | {| | ||
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- | |width=" | + | |width="925px" style="padding: 0 20px 0 0; background-color:#ede8e2"| |
__NOTOC__ | __NOTOC__ | ||
+ | ='''Notebook Microscopy'''= | ||
+ | == Contents == | ||
- | + | {| class="wikitable centered" border="2" rules="rows" width="925px" style="border-color:white;" | |
- | + | |- | |
+ | ! Week !! colspan="7" |Days | ||
+ | |- | ||
+ | |style="text-align:center"| 35 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#8-28-2009|8-28-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#8-31-2009|8-31-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 36 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-02-2009|9-02-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-05-2009|9-05-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-06-2009|9-06-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-07-2009|9-07-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 37 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-09-2009|9-09-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-10-2009|9-10-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 38 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-15-2009|9-15-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-16-2009|9-16-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-20-2009|9-20-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 39 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#9-21-2009|9-21-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 41 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#10-05-2009|10-05-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#10-06-2009|10-06-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 42 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#10-12-2009|10-12-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_measure/NotebookMi#10-13-2009|10-13-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |} | ||
== 8-28-2009 == | == 8-28-2009 == | ||
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[[image:HD09_Hannah_HeLa_GFP.JPG|350px| center|thumb|'''Figure 1: HeLa cells transfected with CMV in front of GFP.''' Image taken with 40x magification.]] | [[image:HD09_Hannah_HeLa_GFP.JPG|350px| center|thumb|'''Figure 1: HeLa cells transfected with CMV in front of GFP.''' Image taken with 40x magification.]] | ||
- | == 9- | + | == 9-02-2009 == |
* Microscopy of cells transfected with cmv, jet, cmv-jet with GFP and an mCherry control | * Microscopy of cells transfected with cmv, jet, cmv-jet with GFP and an mCherry control | ||
* Measurement of exposure time versus average grey value to check whether there is a linear correlation between the two. This measurement was performed using a fluorescent slide that has constant fluorescence and doesn't bleach. The correlation factor was calculated to be 0.9998. Therefore we can calculate the grey values of samples with different exposure times. | * Measurement of exposure time versus average grey value to check whether there is a linear correlation between the two. This measurement was performed using a fluorescent slide that has constant fluorescence and doesn't bleach. The correlation factor was calculated to be 0.9998. Therefore we can calculate the grey values of samples with different exposure times. | ||
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[[image:HD09_microscopy_exposuretime.PNG|center|350px|thumb|'''Figure 2: Graph of exposure time versus average grey values''' The correlation factor is indicated on the diagram.]] | [[image:HD09_microscopy_exposuretime.PNG|center|350px|thumb|'''Figure 2: Graph of exposure time versus average grey values''' The correlation factor is indicated on the diagram.]] | ||
- | == 9- | + | == 9-05-2009 == |
* Test the bleaching effect of our GFP by exciting it over 10 minutes and taking images every 10 seconds. The images were evaluated for their fluorescence intensities using the imaging software NTS Elements-3.1. The data is shown in the diagram below. | * Test the bleaching effect of our GFP by exciting it over 10 minutes and taking images every 10 seconds. The images were evaluated for their fluorescence intensities using the imaging software NTS Elements-3.1. The data is shown in the diagram below. | ||
[[image:HD09_microscopy_bleachingGFP.PNG|center|350px|thumb|'''Figure 3: Bleaching of GFP.''' This diagram shows the percent fluorescence intensity versus time of our GFP. The initial fluorescence intensity was set to 100% The correlation factor is indicated on the diagram.]] | [[image:HD09_microscopy_bleachingGFP.PNG|center|350px|thumb|'''Figure 3: Bleaching of GFP.''' This diagram shows the percent fluorescence intensity versus time of our GFP. The initial fluorescence intensity was set to 100% The correlation factor is indicated on the diagram.]] | ||
- | == 9- | + | == 9-06-2009 == |
* Fixing cells with 4% formaldehyde for standard promoters JeT, JeT-CMV and CMV in HeLa. HeLa cells were cotransected with a plasmid containing JeT in front of mCherry as a reference in a 2:1 GFP: mCherry ratio. | * Fixing cells with 4% formaldehyde for standard promoters JeT, JeT-CMV and CMV in HeLa. HeLa cells were cotransected with a plasmid containing JeT in front of mCherry as a reference in a 2:1 GFP: mCherry ratio. | ||
- | == 9- | + | == 9-07-2009 == |
* Imaging of HeLa with three standard promoters, example image shown below. | * Imaging of HeLa with three standard promoters, example image shown below. | ||
* Image analysis using ImageJ. | * Image analysis using ImageJ. | ||
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[[Image:HD09 microscopy HeLastand.PNG|center|350px|thumb|'''Figure 5: Graph of exposure time versus average grey values''' Mean grey values were obtained using ImageJ analysis of 20x widefield microscopy images. The promoter strength is given relative to our standard promoter JeT. Mean grey values were adjusted according to their exposure time.]] | [[Image:HD09 microscopy HeLastand.PNG|center|350px|thumb|'''Figure 5: Graph of exposure time versus average grey values''' Mean grey values were obtained using ImageJ analysis of 20x widefield microscopy images. The promoter strength is given relative to our standard promoter JeT. Mean grey values were adjusted according to their exposure time.]] | ||
- | == 9- | + | == 9-09-2009 == |
* Fix MCF7 cells for standard measurement. | * Fix MCF7 cells for standard measurement. | ||
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* Imaging and analysis using ImageJ | * Imaging and analysis using ImageJ | ||
- | [[Image:HD09 microscopy MCF7 L4 S4.PNG|center|350px|thumb|'''Figure | + | [[Image:HD09 microscopy MCF7 L4 S4.PNG|center|350px|thumb|'''Figure 7: Relatuve Expression Units of constitutive promoters L4 and S4 compared to standrad promoters JeT-CMV and CMV.''' Mean grey values were obtained using ImageJ analysis of 20x widefield microscopy images. Grey values were adjusted according to their exposure time.]] |
== 9-20-2009 == | == 9-20-2009 == | ||
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* U2OS cell were mostly dead, samples were not measured. | * U2OS cell were mostly dead, samples were not measured. | ||
- | [[Image:HD09 microscopy const060909.PNG|center|350px|thumb|'''Figure | + | [[Image:HD09 microscopy const060909.PNG|center|350px|thumb|'''Figure 8: REUs of constitutive promoters in HeLa.''' Mean grey values were obtained using ImageJ analysis of 20x widefield microscopy images. The promoter strength is given relative to our standard promoter JeT. Mean grey values were adjusted according to their exposure time.]] |
== 10-05-2009 == | == 10-05-2009 == | ||
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== 10-06-2009 == | == 10-06-2009 == | ||
* Imaging and ImageJ analysis of three time-points measurement of induced and uninduced NF-kB clone 31 with P31 (induced and uninduced) as a reference. | * Imaging and ImageJ analysis of three time-points measurement of induced and uninduced NF-kB clone 31 with P31 (induced and uninduced) as a reference. | ||
+ | * Microscopy of plasma membrane targeted GFP (Figure 10). | ||
+ | |||
+ | [[Image:HD09 microscopy NIIL10timepoints.PNG|center|350px|thumb|'''Figure 9: REUs of NIIL10 promoter at three time-points in U2-OS''' U2-OS cells were transfected and fixed after 1, 7 and 10 hours, fixed and measrued the next day. Fluorescence intensity relative to JeT were obtained using image analysis software ImageJ.]] | ||
+ | |||
+ | [[Image:061009 Micha PM green.png|center|350px|thumb|'''Figure 10: Plasma membrane targeted GFP.''' Widefield microscopy image of plasma membrane targeted GFP with 40x magnification.]] | ||
+ | |||
+ | == 10-12-2009 == | ||
+ | * Fixing of U2-OS for standard measurement with CMV, JeT and JeT-CMV and the mCherry reference plasmid. | ||
+ | |||
+ | == 10-13-2009 == | ||
+ | * Imaging of U2OS standards. The exposure times for CMV, JeT and JeT-CMV were 10 ms, 60 ms and 100 ms. | ||
+ | * Image analysis using imageJ was used to measure the flourescence strength. The mean grey values were all normalized to an exposure time of 100ms and the ratio relative to JeT was determined to be: 10.1: 1.0: 0.66 for CMV: JeT :JeT-CMV. | ||
- | [[Image: | + | == 10-21-2009== |
+ | * Imaging of p5_mCHerry_PM [[Image:Measure cherry PM.PNG|center|300px|thumb|'''Figure 11: HeLa cells transfected with p5_mCHerry_PM.''' Imaged 24 h after transfection]] |
Latest revision as of 00:24, 22 October 2009
Notebook MicroscopyContents
8-28-2009
8-31-2009
9-02-2009
9-05-2009
9-06-2009
9-07-2009
9-09-2009
9-10-2009
9-15-2009
9-16-2009
9-20-2009
9-21-2009
10-05-2009
10-06-2009
10-12-2009
10-13-2009
10-21-2009
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