I am currently writing my first master project. I have several images of tissue stained with Alexa Fluor 488, and I need to determine difference in fluorescence intensity between my control and sample group.
The image files are .TIFF.
Huge thanks in advance!
There are programs that can do this. I believe that R has the ability to spot-compare color or brightness in images.
ImageJ software should do a good job in quantifying fluorescence intensities.
Additional information:
https://www.unige.ch/medecine/bioimaging/files/1914/1208/6000/Quantification.pdf
If your images are not 100% covered with tissue, you would first need to select one or more areas in the image where you want to measure intesity (measuring intensity from whole image with varying areas of tissue/withou tissue would make the measurement meaningless). I do not recommend doing this manualy as it will inevitably introduce errors. There are many ways how to do this kind of segmentation, probably the most simple one is thresholding that simply cuts off the background and allows you to measure only useful signal. If you have brightfield images as well, you can use these to select the whole area of tissue, even the parts with no fluorescent signal, but exclude empty areas in image. I would recommend to look at ImageJ and Cellprofiler programs - both require some time to get use to them but they are extremely useful for all different kinds of image analysis.
As a result you will get many statistical parameters describing your images - most of them would probably not be useful for your purpose. Probably the most common parameters used for quantifying fluorescence are mean intensity (simply the average) and integrated fluorescence density (the total amnout of signal). Check this topic, it may help you decide which would be better for your project:
https://www.researchgate.net/post/Is_integrated_density_or_mean_grey_value_better_to_quantify_fluorescence2
Good luck!
Thanks for all the inputs!
I used ImageJ as suggested.
If anyone sees this question later, looking for help, here is my Excel sheet.
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