Dear All:
I wanted to quantify the fluorescence intensity of the images taken by fluorescence microscopy. This can be performed by using ImageJ software. I have attached an image (TIF file) here as well. If I select the whole region of this image and I find mean gray value, does it give the fluorescence intensity of the image? I just wanted to compare the fluorescence intensity between control and experimental groups. I would appreciate it if anyone has any suggestions regarding quantifying fluorescence intensity using ImageJ software.
Thanks,
Nil Pandey
Hi there Nil,
Well, you shouldn't take the fluorescence of the whole picture (is meaningless), but of your regions of interest (ROIs) which in this case are your cells.
Since your image looks pretty clean (high signal-to-noise ratio) do basically the following (maybe you want to modify things afterwards, but this would be the basic workflow):
1) Image>Duplicate...
2) Over the duplicated image:
Image>Adjust>Threshold
-Click Auto*1
-Click Apply
Here you will get a binary image (white and black pixels only)
3)Process>Binary>Watershed (This will split in two cells that are too close and were processed as a single particle)
4)Analyze>Analyze Particles...
(This is the manual: https://imagej.nih.gov/ij/docs/menus/analyze.html#ap)
I would use these settings in your image
-Size: 0-Infinnity
-Circularity: 0.00-1.00
-Check the checkbox: Add to manager
5)Save the ROI manager (it's saved by default as a *.zip file)
6)Click over ("activate") your original image.
-In analyze>Set measurements... Click on the parameters you want to analyze (like Area, IntDensity, Mean Gray Value, etc)
7) In the ROI manager click "Measure"
8) Repeat for the different images.
This will give you the results in the results sheet, copy-paste and that will be it.
*1) Maybe you want to use the same threshold for all your images
Cheeers,
J
Hi there Nil,
Well, you shouldn't take the fluorescence of the whole picture (is meaningless), but of your regions of interest (ROIs) which in this case are your cells.
Since your image looks pretty clean (high signal-to-noise ratio) do basically the following (maybe you want to modify things afterwards, but this would be the basic workflow):
1) Image>Duplicate...
2) Over the duplicated image:
Image>Adjust>Threshold
-Click Auto*1
-Click Apply
Here you will get a binary image (white and black pixels only)
3)Process>Binary>Watershed (This will split in two cells that are too close and were processed as a single particle)
4)Analyze>Analyze Particles...
(This is the manual: https://imagej.nih.gov/ij/docs/menus/analyze.html#ap)
I would use these settings in your image
-Size: 0-Infinnity
-Circularity: 0.00-1.00
-Check the checkbox: Add to manager
5)Save the ROI manager (it's saved by default as a *.zip file)
6)Click over ("activate") your original image.
-In analyze>Set measurements... Click on the parameters you want to analyze (like Area, IntDensity, Mean Gray Value, etc)
7) In the ROI manager click "Measure"
8) Repeat for the different images.
This will give you the results in the results sheet, copy-paste and that will be it.
*1) Maybe you want to use the same threshold for all your images
Cheeers,
J
Dear Dr. J. Ramirez-Franco :
Thank you for your help. I really appreciate it. I just wanted to quantify the fluorescence intensity, and I will follow your steps. I have a follow-up question on your answer. At this time, my question is: does ‘Mean gray value’ give fluorescence intensity, or do I need to determine other parameters as well (such as area and integrated density) to know fluorescence intensity?
Thanks,
Nil Pandey
Hi there Nil Kanatha Pandey ,
Glad to help. We just discussed this question in the RG forum a couple of weeks ago, please follow the link below which, in turn, provides more useful links in this regard.
https://www.researchgate.net/post/Mean_gray_intensity_value_or_the_integrated_density_value_which_among_the_two_value_is_more_appropriate_for_quantifying_the_fluorescence_image_data
Cheers,
J
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