fluorescence intensity

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fluorescence intensity

Anette Christ
Dear ImageJ,
I have a question how to use the ImageJ program. I have labeled cells with a fluorescence dye. I let the cells in culture about several days. Than I made pictures with the fluorescence microscope on day 1, 3 and 5 after staining. Now I want to messure and compare the fluorescence intensity signal over these days and furthermore I want to examine whether there is any weakening.
So, can you say me how to to this with the ImageJ program?

Thank you.

With kind regards,
Anette Christ


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Re: fluorescence intensity

Michael Weber-4
Hi Anette,

the are a couple of ways to measure intensity. The most simple way is to
load an image, point the mouse to the pixel of interest and check the
signal intensity in the main tool bar. This could be a first test if the
images where acquired in the range of the detector (=if nothing it
overexposed). Did you acquire all images under the same condition
(excitation power, filter setup, exposure time)? Otherwise it will be
tricky to compare them.

You can also measure regions by drawing i.e. a circle in the cell and go
for "Analyze" > "Measurements". There are also plugins/macros to automate
this, but a couple of manual steps before are probably go to find the best
way.

cheers,
Michael


> Dear ImageJ,
> I have a question how to use the ImageJ program. I have labeled cells with
> a fluorescence dye. I let the cells in culture about several days. Than I
> made pictures with the fluorescence microscope on day 1, 3 and 5 after
> staining. Now I want to messure and compare the fluorescence intensity
> signal over these days and furthermore I want to examine whether there is
> any weakening.
> So, can you say me how to to this with the ImageJ program?
>
> Thank you.
>
> With kind regards,
> Anette Christ
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Re: fluorescence intensity

Joel Sheffield
I would emphasize Michael's point about the range of intensity.  You
must make sure that your brightest image is within the 0-255 range of
the 8-bit image (for example).  You will not be able to compare two
images each of which is so bright that it exceeds that limit.

Joel

Date sent:       Mon, 24 Sep 2007 11:54:13 +0200
Send reply to:   [hidden email]
From:           Michael Weber <[hidden email]>
Subject:         Re: fluorescence intensity
To:             [hidden email]

> Hi Anette,
>
> the are a couple of ways to measure intensity. The most simple way is to
> load an image, point the mouse to the pixel of interest and check the
> signal intensity in the main tool bar. This could be a first test if the
> images where acquired in the range of the detector (=if nothing it
> overexposed). Did you acquire all images under the same condition
> (excitation power, filter setup, exposure time)? Otherwise it will be
> tricky to compare them.
>
> You can also measure regions by drawing i.e. a circle in the cell and go
> for "Analyze" > "Measurements". There are also plugins/macros to automate
> this, but a couple of manual steps before are probably go to find the best
> way.
>
> cheers,
> Michael
>
>
> > Dear ImageJ,
> > I have a question how to use the ImageJ program. I have labeled cells with
> > a fluorescence dye. I let the cells in culture about several days. Than I
> > made pictures with the fluorescence microscope on day 1, 3 and 5 after
> > staining. Now I want to messure and compare the fluorescence intensity
> > signal over these days and furthermore I want to examine whether there is
> > any weakening.
> > So, can you say me how to to this with the ImageJ program?
> >
> > Thank you.
> >
> > With kind regards,
> > Anette Christ


--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]  
(215) 204 8839, fax (215) 204 0486
http://astro.temple.edu/~jbs