Fluorescence intensity measurement

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Fluorescence intensity measurement

María Victoria Pepe
Hello

I am analyzing the expression of gfp in a group of bacteria.My total
population is marked with an antibody (channel 1) and some bacteria express
gfp with different intensities (channel 2)
I want to calculate the fluorescence intensity for each bacterium, That is,
for the total number of bacteria (which I see on channel 1) I want to
calculate the fluorescence intensity on channel 2
My antibody is extracellular and gfp is intracellular

What would be the best tool??

Thanks a lot

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Re: Fluorescence intensity measurement

G. Esteban Fernandez
Hi Maria,

You will use the Analyze Particles tool, but you need to do several steps
to prepare your images to get a good result.

1. Split your two channels into two windows using Image > Color > Split. If
they are already saved in two different files then you don't have to split,
just open the two images so that they are in two different windows.

2. Run Analyze > Set Measurements and check the box for Mean intensity.
Also select the channel 2 image name in the "Redirect to" drop-down; this
is so intensity measurements are done in channel 2.

3. Select the channel 1 window and run Image > Adjust > Threshold. This is
to set channel 1 as the basis for the objects of interest.

3. Select "Red" from the drop-down in the Threshold window and adjust the
slidebars until your bacteria are completely highlighted in red but no
background is highlighted. If tiny background artifacts are highlighted
that's OK, you can filter them out later.
If it's not possible to highlight/threshold only the bacteria without
background then reset/clear the threshold and run Process > Subtract
Background first, then try Threshold again. If it STILL doesn't work then
this gets more complicated and you have to get fancy with the thresholding,
e.g. Auto Local Threshold in Fiji ImageJ.

4. Once your bacteria are highlighted/thresholded in channel 1, with the
channel 1 window activated run Analyze > Analyze Particles. The objects
will be detected in channel 1 but intensity will be measured in channel 2,
as you set in step 2 above.

5. Check the boxes for Clear results and Show results.  Select "Outlines"
from the "Show" drop-down to see the objects quantified. If there are tiny
artifacts to filter out then type in a minimum size for objects of interest
in the Size box (default is 0.0; leave the max. as Infinity unless there
are also large artifacts).

6. Click OK in the Analyze Particles window. An table with the results will
pop up and so will an image map of the objects that were quantified.

Good luck and let us know if you need anything else!

-Esteban


On Jun 5, 2017 10:21 AM, "María Victoria Pepe" <[hidden email]>
wrote:

Hello

I am analyzing the expression of gfp in a group of bacteria.My total
population is marked with an antibody (channel 1) and some bacteria express
gfp with different intensities (channel 2)
I want to calculate the fluorescence intensity for each bacterium, That is,
for the total number of bacteria (which I see on channel 1) I want to
calculate the fluorescence intensity on channel 2
My antibody is extracellular and gfp is intracellular

What would be the best tool??

Thanks a lot

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html