Hi,
I'm currently working on the dimerization of two solute carriers. For that I'm using bi molecular fluorescence complementation. I've transfected 293FT cells attached to poly-D-lysine coated coverslips, with my constructs (including a positive control). The signal from the complementation of the solute carriers is not as strong as the positive control, which is anticipated, but it is also not that much stronger than my background fluorescence. I'm using Fiji/ImageJ to analyze my pictures. However, I have some trouble figuring out how to go about it. How do I subtract the background fluorescence and how do the threshold function work?
Thanks
Hello,
ImageJ/Fiji (that's ImageJ but with a lot more Plugins added, you should check it out) is perfectly suited for the task you describe. You have several options to subtract the background and apply a threshold. However, to go into detail, it would be helpful to have an example image at hand. Could you please attach one (not as jpeg, but as tif!) and describe in more detail which data you want to obtain from it? That would be great.
Regards,
Leonhard
Hi Leonhard,
Thank you for your reply. I'm sorry for not writing back sooner. I've attached two images of my cells. The one named positive is the one with my weak signal (generated by complementation of two proteins tagged with the halves of venus YFP, residues 1-173 and 155-238). The negative is cells transfected with an empty vector. As you can see, the signal is weak, however, it is there. What I would like is to use the information from the negative image to subtract the autofluorescence from the positive image in order to accentuate the signal from the complementation.
Again, thank you very much for taking your time to help me.
Regards,
Frederik
Dear Frederik,
thanks for the images. As far as I see, they are RGB-images originating from two channels, green and yellow. I suppose the yellow channel contains your signal. However, with the overlay, it is difficult (albeit not impossible) to obtain the information you need. Therefore, if you want to use these images to quantify your signal, I would sggest you have a look at only the channel where the signal is present. Usually, you can set this when storing the images in the microscope software: Store each channel seperately as a 16-bit tif. Then you have an image with pixel-by-pixel grey values that you can easily use to quantify the signal.
The idea for the quantification is the following: Measure the signal you obtain in the negative control. You can then apply this value as threshold for the positive control or subtract it. This depends a little on the experiment. It is, however, important to use one method throughout one analysis and not change it. I will show it to you in more detail.
By the way: Which microscope do you use and which magnification? If the size of the cells in the images is typical, the magnification may be too low to obtain enough photons from you venus construct. It could well be that the signal will increase just by using a higher magnification (63x or 100x objective, for example).
Regards,
Leonhard
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