Color Merge Multiple Fluorescence Images

[Breukers, C (TNW)]

Dear all,

 

I'm working on a fluorescence microscope that has four fluorescence
channels (DAPI, FITC, PE, APC).

 

Since we are measuring antibody conjugated fluorescent cells, we have a
large variation in intensity and background.

 

What would be the correct way to have a color merge of four fluorescence
microscope images / channels?

What pre processing steps are necessary to modify the images such that a
nice clear color overlay is created?

 

Kind regards,

Christian Breukers

---------------------------------------------------
University of Twente

Faculty of Sciences and Technology

MIRA Institute for Biomedical Technology and Technical Medicine

Department of Medical Cell BioPhysics
---------------------------------------------------

 

[John Oreopoulos]

See color submenu here:

http://rsbweb.nih.gov/ij/docs/menus/image.html#color

RGB Merge
Merges 1-4 greyscale images or stacks into an RGB image or stack.  
Select *None* to keep a channel empty (filled with 0). Check "Create  
Composite" to convert 2-4 grayscale images or stacks into a composite  
image or hyperstack. Check "Keep Source Images" if you wish to keep  
the originals.

Adjust the brightness and contrast of your channel images first (and  
make sure you say that you have done this in your figure captions or  
your methods sections! - See Rossner, M., and K. M. Yamada. 2004.  
What's in a picture? The temptation of image manipulation. J. Cell  
Biol. 166:11-15). Then run the RGB merge command. If you have many  
image sets to do this on, you can automate by writing a simple macro  
in ImageJ

John Oreopoulos


On 21-Jan-10, at 3:58 AM, Christian Breukers wrote:

> Dear all,
>
>
>
> I'm working on a fluorescence microscope that has four fluorescence
> channels (DAPI, FITC, PE, APC).
>
>
>
> Since we are measuring antibody conjugated fluorescent cells, we  
> have a
> large variation in intensity and background.
>
>
>
> What would be the correct way to have a color merge of four  
> fluorescence
> microscope images / channels?
>
> What pre processing steps are necessary to modify the images such  
> that a
> nice clear color overlay is created?
>
>
>
> Kind regards,
>
> Christian Breukers
>
> ---------------------------------------------------
> University of Twente
>
> Faculty of Sciences and Technology
>
> MIRA Institute for Biomedical Technology and Technical Medicine
>
> Department of Medical Cell BioPhysics
> ---------------------------------------------------
>
>

[Daniel James White]

Dear Christian,

see bwlow,

Begin forwarded message:

> Date:    Thu, 21 Jan 2010 10:54:53 -0500
> From:    John Oreopoulos <[hidden email]>
> Subject: Re: Color Merge Multiple Fluorescence Images
>
> See color submenu here:
>
> http://rsbweb.nih.gov/ij/docs/menus/image.html#color
>
> RGB Merge
> Merges 1-4 greyscale images or stacks into an RGB image or stack.
> Select *None* to keep a channel empty (filled with 0). Check "Create
> Composite" to convert 2-4 grayscale images or stacks into a composite
> image or hyperstack. Check "Keep Source Images" if you wish to keep
> the originals.
>
> Adjust the brightness and contrast of your channel images first (and
> make sure you say that you have done this in your figure captions or
> your methods sections! - See Rossner, M., and K. M. Yamada. 2004.
> What's in a picture? The temptation of image manipulation. J. Cell
> Biol. 166:11-15). Then run the RGB merge command. If you have many
> image sets to do this on, you can automate by writing a simple macro
> in ImageJ
>
> John Oreopoulos

You can do it like that for sure!

But remember, there are only 3 proimaty colours.
So where you have R, G and B strong in the same place,
you wil see white,
and you cant distinguish that from the 4th grey scvale channel!!!

your merge images might be impossible to interpret sensibly unless the  
spatial location of all 4 dyes is well separated.

Dan

>
>
> On 21-Jan-10, at 3:58 AM, Christian Breukers wrote:
>
>> Dear all,
>>
>>
>>
>> I'm working on a fluorescence microscope that has four fluorescence
>> channels (DAPI, FITC, PE, APC).
>>
>>
>>
>> Since we are measuring antibody conjugated fluorescent cells, we
>> have a
>> large variation in intensity and background.
>>
>>
>>
>> What would be the correct way to have a color merge of four
>> fluorescence
>> microscope images / channels?
>>
>> What pre processing steps are necessary to modify the images such
>> that a
>> nice clear color overlay is created?
>>
>>
>>
>> Kind regards,
>>
>> Christian Breukers
>>
>> ---------------------------------------------------
>> University of Twente
>>
>> Faculty of Sciences and Technology
>>
>> MIRA Institute for Biomedical Technology and Technical Medicine
>>
>> Department of Medical Cell BioPhysics
>> ---------------------------------------------------
>>
>>
>

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )