I have stain my retinal slides with GFAP. I am trying to quantify the intensity of the gfap signal using image j. Currently the only method I able to find is calculating using corrected total cell fluorescence. Is there any suggestion or any other recommendation?
Hi Fu Shun Wong,
I am also using Image J for quantifying the fluorescence. What I am doing is that I subtract the background from the images. Then I mark the defined area in which I want to quantify the fluorescence and add that area in ROI manager. Then I do threshold of the image and then click on the ROI in ROI manager and then click measure in the Analyze option. Then a new window opens with the result of the measured intensity.
I hope this helps.
Regards,
Ekta
Hi Ekta,
Thanks for replying, I will try your method.
What I did was, I convert my images to grayscale first. Then I select the area I want to measure, including an area of the background. Then I use the formula "integrated density of selected area - (area of selected area * mean fluorescence background)". Is this method acceptable?
hi Fu Shun Wong,
if you are trying to quantify any signal from microscopy images, you have to take care of several parameters.
1. flat field correction 2. control slides 3. presence of IR filter to camera 4. finding linearity of your system. 5. stability of your light source of microscope throughout experiment. and some other controls to make sure your microscope is working well for quantitation.
for your question regarding image analysis following links would help you to quantify signal from your images.
https://digital.bsd.uchicago.edu/resources_files/Basic%20image%20quantification.pdf
http://sciencetechblog.com/2011/05/24/measuring-cell-fluorescence-using-imagej/
make sure that you have't saturated image while grabbing and using flat field correction. if you have taken image with color camera and now converting it to black n white doest give good results. and images that are not corrected for flat field can change intensity of similar intensities upto 40%.
all the best :)
Hi Fu Shun Wong,
The integrated density is equivalent to the product of area selected and its mean gray value. So, it is not the real quantification of the fluorescence. If you want to measure the real values of the fluorescence and want to compare between a test group and control group then I think the method that I am using is optimum. I am using it for comparing fluorescence intensities between KO and WT cells.
Regards,
Ekta
Hi Ekta,
when you subtract your background, how much pixels you subtract? But under seteasurent there is only integrated density. I am comparing GFAP intensity between normal and damage retina..
Hi Fu Shun Wong,
I first select a background area (a small square from the background), add in ROI manager and then subtract it from the image. For this you need a plugin in ImageJ called Background Subtraction. In the Analyze option there is Set Measurements. There I tick the box for Area, Mean gray value, Limit to threshold and Display label. Then I go to my image, click on it and open the Measure option from the Analyze option. Then it give out the values.
Regards,
Ekta
Hello Ekta
I have a similar question and I wanted to know in detail how you do the measurement of fluorescence. Where can I find the plugin you mentioned? Also once I get the values , how do I calculate the fluorescence from each cell in the field supposing I have many cells in one single field with few cells showing fluorescence and few without.
Thank you very much
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