Team:Heidelberg/Notebook measure/NotebookMi

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=Notebook Microscopy=

8-28-2009

 * Fixing cells with 4% Formaldehyde for microscopy on monday.

8-31-2009

 * Microscopy of our first fixed cells transfected with various constructs at the Nikon imaging center
 * Cells transfected with cmv-GFP, jet-GFP, cmv-jet-GFP
 * The figure below shows HeLa cells transfected with GFP under a cmv promoter. The image was taken using the Nikon Eclipse 90i upright automated widefield microscope with 40X magnification:



9-02-2009

 * 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.



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.



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.

9-07-2009

 * Imaging of HeLa with three standard promoters, example image shown below.
 * Image analysis using ImageJ.





9-09-2009

 * Fix MCF7 cells for standard measurement.

9-10-2009

 * Imaging and analysis of MCF-7 with JeT, JeT-CMV and CMV.



9-15-2009

 * Fix MCF-7 cells for testing constitutive promoter L4 and S4

9-16-2009

 * Imaging and analysis using ImageJ



9-20-2009

 * Fix HeLa and U2OS cells for testing constitutive promoters S5, S10, S16, L1, L8 and JeT as reference.

9-21-2009

 * Imaging and Analysis of fixed HeLa with constitutive promoters.
 * U2OS cell were mostly dead, samples were not measured.



10-05-2009

 * Fixing U2-OS cells transfected with NF-kB clone 31 inducible synthetic promoter at three different time points. Cells were induced at 7:30 am. Cells were fixed after 3, 7 and 10 hours.

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.
 * Microscopy of plasma membrane targeted GFP (Figure 10).





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.

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]]