Hello!
I am currently working on FRET using acceptor photobleaching. I have obtained images from a Zeiss 880 confocal. Using the ROI feature, I am able to calculate a 15-30% increase in the donor channel once the acceptor is bleached. However, when I use imageJ to use the image calculator, it is unable to pick up the donor increase. Any plugin suggestions and/or other analyzing methods?
Thank you in advanced.
Hi Claira, maybe an example image and more details of the analysis steps you are taking in Image J will help to understand better what is the issue. It's not very clear to me why you are using image calculator, what operation you do in the calculator and what information you expect from it. Do you want to get an image of the donor channel in which the region, where acceptor has been bleached, appears brighter? It may not be that obvious if your image is noisy and the features are sparse. It may be better just to take the average intensities from the region, which as you said showed a difference
Radek Machan thank you for this advice. As for ImageJ, I am using the image calculator to first calculate the mean and SD of the area where the donor should have an increase mTFP1 intensity and outside of that to make sure it is just my region of interest that is getting affected by the bleaching. Also, I do it with the acceptor as well to make sure I do not see any changes between pre and post bleach images. When I use the image calculator, I divide the post image by the pre image and increase the brightness until I am able to see the region that was bleached.
Dividing one image by another can be tricky as the noise may become quite high (in background areas you divide one small number by another, which can give higher numbers than in the areas with signal). Have you tried to subtract the images?
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