Hello,
i already measured the fluorescence intensity of a neuropeptide using integrated optical density with ImageJ.
The aim: to figure out the fluorescence intensity of only one neuropeptide of 2 different latency periods after treatment (mouse eyes) and compare with the control group.
I set a constant threshold and then measure the specific area, using a template (set measurements: integrated density etc.). Is that the right approach to measure fluorescence intensity of paraffin slides?
Are there any other methods to measure the fluorescence??
For a better understandig i added a sample image (mouse retina; 20x; stained with a neuropeptide)
Thank you!
I think you have done what needs to be done to measure fluorescent intensity. it is hard to measure protein in the tissue due to the lack of structural components that we can use to identify what we are measuring. In my work i used to break down the tissue into segments of the same size. measure each segment's intensity and then collect the data as separate readings of the intensity in different parts of the image. This was quite useful when measuring CGRP intensity in the colon. each crypt was a segment for me. each image carried between 20-30 crypts. subsequently i would get 20-30 readings and then would take the SEM of each image and plot it out on Prism. This narrows down the noise.
good luck
I just saw this! i apologize!
in short yes!
Identify the intensity of the control, then subtract that from the intensity of your stained samples
to make it more accurate don't do an overall measurement of your single image. break your image into sections (1cmx1cm) and use them as separate readings for a single image. Then you will be taking account the normal variation you see in any stained section (the intensity of the colour and the depth of the staining varies between different areas of the image)
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