Re: Florescence Intensity

[AJBell]

Sam,

Speaking from personal experience, I can honestly say it may not be
possible due to small variations between the images. That being said, the
method I used to calculate intensity per cell was to threshold the image
automatically, create selection, analyse particles then take measurements
of cell size and Integrated density.

This can be put into a macro quite easily.

Hope that helps.

Andrew


On Fri, 10 Apr 2009 14:11:46 +0100, Sam Murray
<[hidden email]> wrote:

>Hello.  I was wondering whether someone could help me please. I am
looking at the effect of growing brain cells in 3 different sera (human,
serum free and fetal calf). I would like to see the effect of the sera on
8 different antibodies by flow cytometry and immunocytochemistry. The flow
cytometry analysis is complete, I would now like to compare the ICC. All
micrographs have been taken with a b&w camera and all with the same
settings. How can I use ImageJ to quantify the fluorescence intensity? I
probably wouldn't need to subtract the background as I have appropriate
background ICCs. I have around 200 micrographs, so some sort of
automation/plugin would be good.
>
>Your help is very much appreciated.
>Kind regards,
>Sam
>========================================================================

[dpoburko]

Hi Sam,
 
   There is a great example of how to write a batch macro that can step
through folders and subfolders using a filter to select specific images
for anlysis (or in the case of the macro to build a stack). See
BatchProcessFolders.txt in the examples. We are doing something similar
to what you want to do. We are using a defined grey value for
thresholding, as like in your case all images were acquired at the same
setting. Here is the outline of our algorythm:
Make sure you have a logical naming system for you images. (i.e.
something like YYMMDD_condition_Slide_Cell_color_Z)
Using the batch macro to work through the images
    (For example, put various treatment groups in folders, where the
folder name matches the basename of the images to be analyzed)
   
    Nest for loops work through various colors, etc
       Gaussian Blur (~0.9 sigma, smooths speckles in the thresholded image)
              SetThreshold (probably with a fixed value for all images)
                    Analyze Particles (set size and circularity to
restrict counting to roughyl soma sized objects)
                          Save measurements to a txt file named
according to the current image

This should allow you to batch process all of your images in less than
an hour. If you would like an example macro (albeit a messey), just let
me know.

Cheers,
Damon



Andrew James Bell wrote:

> Sam,
>
> Speaking from personal experience, I can honestly say it may not be
> possible due to small variations between the images. That being said, the
> method I used to calculate intensity per cell was to threshold the image
> automatically, create selection, analyse particles then take measurements
> of cell size and Integrated density.
>
> This can be put into a macro quite easily.
>
> Hope that helps.
>
> Andrew
>
>
> On Fri, 10 Apr 2009 14:11:46 +0100, Sam Murray
> <[hidden email]> wrote:
>
>  
>> Hello.  I was wondering whether someone could help me please. I am
>>    
> looking at the effect of growing brain cells in 3 different sera (human,
> serum free and fetal calf). I would like to see the effect of the sera on
> 8 different antibodies by flow cytometry and immunocytochemistry. The flow
> cytometry analysis is complete, I would now like to compare the ICC. All
> micrographs have been taken with a b&w camera and all with the same
> settings. How can I use ImageJ to quantify the fluorescence intensity? I
> probably wouldn't need to subtract the background as I have appropriate
> background ICCs. I have around 200 micrographs, so some sort of
> automation/plugin would be good.
>  
>> Your help is very much appreciated.
>> Kind regards,
>> Sam
>> ========================================================================
>>    

--

Damon Poburko, PhD
Postdoctoral Research Fellow
Stanford University School of Medicine
Dept. of Molecular & Cellular Physiology
279 Campus Dr., Beckman B103, Stanford, CA 94305
Ph: 650 725 7564, fax: [phone]