Team:Heidelberg/Notebook measure/NotebookMi

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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:
Figure ?

9-2-2009

  • 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.
Figure ?: Graph of exposure time versus average grey values The .The standard deviations of the replicates are also shown.

9-5-2009

  • 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.
Figure 2: This diagram shows the percent fluorescence intensity versus time of our GFP. The initial fluorescence intensity was set to 100%.

9-6-2009

  • 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.

9-7-2009

  • Imaging of HeLa with three standard promoters, example image shown below.
Figure 3: HeLa cells co-transfected with cmv-GFP and JeT mCherry both fluorescence proteins are localized to the plasma membrane.
  • Image analysis using ImageJ.
Figure 1: Graph of exposure time versus average grey values


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