I am measuring the fluorescence intensity of subcellular organelles (corrected fluorescence) by ImageJ in the same way by which corrected total cellular fluorescence is measured. I want to know if relative intensity is a reliable method. Does the relative intensity differ a lot if the antibodies differ or it remains unaltered since the relative intensity is corrected by background elimination? Also would be helpful if there is any published material.
You can only use intensity as a metric if your samples are:
- Prepared at the same time, in parallel
- Prepared with the exact same procedure
- Imaged at the same time, in sequence (same microscope settings, exposure times, excitation intensities, etc)
- Kept within the most similar type of field quality as possible (background levels, cell numbers, quality of mounting, etc)
And even then, you have to be very careful about not taking liberties with analysis. You can improve the quality if the analysis if you have a protein that should be the same in all conditions and then show that in your sample handling and imaging, the intensity of this target was the same across all conditions. This would be your normalizing factor.
In most cases, it is best to use fixed imaging as a measure of morphology and spatial arrangement, rather than an intensitometric technique. In contrast, with live cell imaging you can take a baseline and then compare the relative change to baseline across different conditions. This pre-treatment baseline is effectively an internal normalization.
Be aware of the difference between average intensity and integrated intensity:
https://www.researchgate.net/post/Is_there_a_written_guideline_for_image_acquisition_and_processing_using_FIJI_ImageJ_for_the_final_goal_of_determining_Mean_Gray_Value_of_entire_image
In our facility we routinely use fluorescence beads (http://www.spherotech.com/flu_par_sli.htm) with linearly increasing intensities to obtain a calibration curve.
I suppose that you are using a confocal microscope here.
In addition to already rich answers, consider that subcellular features are not always in focus. You may have to acquire more that one image, a z-stack that you can process afterwards with aforementioned techniques. It may help you recover the total fluorescence emanating from the organelles that you are studying, but it may need some time to fine-tune the imaging sequence so that it would remain identical from a sample to another.
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