color merge

[Surita Banwait]

Hi There

 

Is there a way to make a color merge of a '5' color image (i.e.
Alexa488, Alexa555, Alexa647, DAPI and then the merged image of the 4
channels)?  The color merge plug in will only give me up to 4 boxes to
choose from and it removes one of my channels in the data set.

 

Thanks,

-Surita

[Surita Banwait]

 

Hi There

 

Is there a way to make a color merge of a '5' color image (i.e.
Alexa488, Alexa555, Alexa647, DAPI and then the merged image of the 4
channels)?  The color merge plug in will only give me up to 4 boxes to
choose from and it removes one of my channels in the data set.

 

Thanks,

-Surita

[lechristophe]

You already sent this very same email two days ago....

There's no such thing as five color merge, that's why nobody replied to
the first mail I guess !
Color space has only three dimension (RGB), four if you add intensity
(like in CMYK), that allows to get greyscale as a 4th "color".
Real four color merge will lead to non-interpretable overlays, beacause
your fourth color will necessarily be a combination of the first three
colors used, don't even try with five.
My advice is that you chose two overlays of three and two colors (let's
say red/green/blue and magenta/cyan),  choosing the combination to show
what's more important in terms of colocalization...

Christophe

Surita Banwait a écrit :

>  
>
> Hi There
>
>  
>
> Is there a way to make a color merge of a '5' color image (i.e.
> Alexa488, Alexa555, Alexa647, DAPI and then the merged image of the 4
> channels)?  The color merge plug in will only give me up to 4 boxes to
> choose from and it removes one of my channels in the data set.
>
>  
>
> Thanks,
>
> -Surita
>
>  

[Joel Sheffield]

Here are two approaches.

You can use the Process>Image Calculator to add images to each other.
 This has to be done as separate operations.  i.e. add 1 and 2 to
yield a result.  Then add 3 to the result of that, then add 4 to the
result of that etc.

You should convert all of your images to rgb before doing the
additions.  Also, you should be aware of possible saturation.  That
is, if the sum of the values for any of the pixels is greater than
255 in any of the channels, you will be beyond the useful range.  I
would recommend using the Process>Math function to divide each of the
images by some number that will reduce the total.  That way you'll be
safe, and can make final adjustments on the resulting image.  You
will, however, lose some bit depth.  If your differrent channels are
non-overlapping, this should not be a problem.


An alternate way to approach this is to
1. convert all images to rgb
2.  make a stack containing all of the images
3.  divide the stack as above (Process>Image math)
4.  use Image>Stacks>Z-project, and select "sum slices"

Joel

>
>
> Hi There
>
>
>
> Is there a way to make a color merge of a '5' color image (i.e.
> Alexa488, Alexa555, Alexa647, DAPI and then the merged image of the 4
> channels)?  The color merge plug in will only give me up to 4 boxes to
> choose from and it removes one of my channels in the data set.
>
>
>
> Thanks,
>
> -Surita

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

[dscho]

Hi,

On Fri, 29 Sep 2006, Christophe Leterrier wrote:

> You already sent this very same email two days ago....

True. I also saw it, and was still thinking about it.

> There's no such thing as five color merge, that's why nobody replied to the
> first mail I guess !

Not exactly true. You _can_ merge with five colours, and if the signals
are mostly non-overlapping, you get sensible results even if mixing
colours.

The only question is: how?

First of all, RGB is an unexact colour space. (See
http://en.wikipedia.org/wiki/Lab_color_space#RGB_and_CMYK_conversions).

Ignoring this fact, let's just say we had n gray value images and n
colours. If we just built the weighted sum per pixel (sum of gray value
times colour value), the sum could overload (i.e. it could yield values
for R, G or B > 255).

Okay, so how about averaging the weighted values (sum of gray value times
colour values, divided by sum of gray values)? This would not match our
intuition: if n=2, at a certain coordinate, one gray value is 255, and the
corresponding colour is green (#00ff00), and the other image's gray value
is also 255, with corresponding colour magenta (#ff00ff), we would expect
the result to be white (#ffffff), right?

I have not searched for any research papers on the subject, but maybe
someone on this list can tell us more about the subject (and a solution)?

Ciao,
Dscho

[Joachim Walter]

Surita,

I agree with Johannes: merging more than 3 colors gives sensible results
when you have non-overlapping signals, but you might have to play with
the settings. A way to do this interactively is with Image5D
(http://rsb.info.nih.gov/ij/plugins/image5d.html).

After installing Image5D, open the five images (I assume you have
images, not image stacks) in ImageJ and merge them to a stack
("Image->Stacks->Convert Images to Stack"). Then select
"Plugins->Image5D->Stack to Image5D". In the dialog box select "ch" as
3rd dimension and "z" or "t" as 4th, "5" as 3rd dimension and "1" as 4th
dimension size, and click "ok". You get a Window displaying you data
with a slider on the right to change the displayed channel.

Click on the "Color" (right up) and you get a palette to choose the
display color, i.e. its hue and saturation, for the currently selected
(displayed) channel. To change the brightness of a channel, use ImageJ's
"Image->Adjust->Brightness/Contrast" Dialog. N.B.: If you have 16-bit
images, an easy way to get a good starting point for the contrast is to
keep the space-bar pressed while changing the channel.

Select your choice of color and adjust the contrast for each channel.
Then, in the dropdown box below the "Color" button, select "ovl". You
will see an "overlay" image, which is simply an RGB-image with the
RGB-values of all channels added up. I think most software for
multi-color image acquisition does it this way, because it's fast. In
overlay mode, the slider on the right is replaced with a control with
one box for each channel that contains a radiobutton (round) and a
checkbox (squared). You can still change the display color and
brightness/contrast in this mode. Any changes act on the channel
selected with the radiobutton. With the checkbox you can toggle the
display of the particular channel.

To get an RGB-image from the displayed overlay, use the
"Plugins->Image5D->Image5D Stack to RGB" plugin. If you have a number of
datasets of the same type you might want to select the display color and
brightness for one dataset and transfer the selected settings to the
other datasets using ""Plugins->Image5D->Transfer Channel Settings".

All the best,
Joachim


Johannes Schindelin schrieb:

> Hi,
>
> On Fri, 29 Sep 2006, Christophe Leterrier wrote:
>
>  
>> You already sent this very same email two days ago....
>>    
>
> True. I also saw it, and was still thinking about it.
>
>  
>> There's no such thing as five color merge, that's why nobody replied to the
>> first mail I guess !
>>    
>
> Not exactly true. You _can_ merge with five colours, and if the signals
> are mostly non-overlapping, you get sensible results even if mixing
> colours.
>
> The only question is: how?
>
> First of all, RGB is an unexact colour space. (See
> http://en.wikipedia.org/wiki/Lab_color_space#RGB_and_CMYK_conversions).
>
> Ignoring this fact, let's just say we had n gray value images and n
> colours. If we just built the weighted sum per pixel (sum of gray value
> times colour value), the sum could overload (i.e. it could yield values
> for R, G or B > 255).
>
> Okay, so how about averaging the weighted values (sum of gray value times
> colour values, divided by sum of gray values)? This would not match our
> intuition: if n=2, at a certain coordinate, one gray value is 255, and the
> corresponding colour is green (#00ff00), and the other image's gray value
> is also 255, with corresponding colour magenta (#ff00ff), we would expect
> the result to be white (#ffffff), right?
>
> I have not searched for any research papers on the subject, but maybe
> someone on this list can tell us more about the subject (and a solution)?
>
> Ciao,
> Dscho
>  

--
------------------------------------------------------------------
  Dr. Joachim Walter
  Till I.D. GmbH
  c\o BioImaging Zentrum
  Großhaderner Str. 2
  82152 Martinsried, Germany

  Tel.: [phone]
  Fax:  [phone]

  [hidden email]
------------------------------------------------------------------

[Herbert M. Geller]

Surita,

You have received lots of comments about how images are only RGB.  This
is true.
The problem is if you have grey scale images that are not the actual
color of the fluorophore.  However, if you capture your images in color,
rather than grey scale, each image will have an RGB component.  You can
then create R, G, and B images from each of the color images, and then
add each of the R, G, and B images together.  You would then convert the
added R, G, and B images into a single RGB image that will have 4 colors
that closely approximate the fluorophores you are using.
If you only have grey scale, then you can make "false" color by
converting them first to indexed color with a LUT, and then pick a color
for the most intense pixel that approximates 488, etc, and then convert
the images to RGB.  Then do this for the others.  I am not sure how easy
it is to do this in ImageJ, but it is straightforward in Photoshop.

Herb Geller


Surita Banwait wrote:

>Hi There
>
>
>
>Is there a way to make a color merge of a '5' color image (i.e.
>Alexa488, Alexa555, Alexa647, DAPI and then the merged image of the 4
>channels)?  The color merge plug in will only give me up to 4 boxes to
>choose from and it removes one of my channels in the data set.
>
>
>
>Thanks,
>
>-Surita
>  
>

--
--------------------------------------
Herbert M. Geller, Ph.D.
Developmental Neurobiology Section
National Heart Lung and Blood Institute, NIH
10 Center Drive MSC 1754
Bldg 10, Room 6D18
Bethesda, MD  20892-1754
Tel: [phone]; Fax: [phone]
e-mail: [hidden email]
Web: http://dir.nhlbi.nih.gov/labs/ldn/index.asp
---------------------------------------

[Surita Banwait]

Hi There

I wanted to thank everyone for all of their suggestions regarding
merging images.  I think I should have made myself a bit more clear...

We have a Zeiss LSM 510 and are capable of scanning up to 4 different
fluorophores.  We have generated several images in which there is a
'green' channel (FITC), 'red' channel (cy3) 'pink' channel (cy5) and
'blue' channel (AMCA/DAPI).  

First, when I try to open these 4 color images using the LSM toolbox,
one of the channels is lost.  I guess I would have to save each channel
separately to get around this.

(I am quite familiar with photoshop and to avoid problems I save each
channel as a grey scale image and then save the RBG channels in the
appropriate channels.  i then create an additional channel to add the
final color; then to save the image, I just merge everything and save as
a jpeg, and for panel shots I can save each channel as a separate
channel).  

I discovered the 'RGB to Montage' option within 'color functions' in the
plugins menu.  This works great for RGB merged images, but I was
wondering if this can be done with the additional color ... it is simple
enough to create a panel with my LSM software, was just curious if
ImageJ could do something similar/ if there was a way to modify this
macro to include an additional channel/ box in the options that pop up
when creating the panel  

Thanks again for the comments,
-Surita




------------------------

[sharmistha paul]

Surita Banwait <[hidden email]> wrote:  Hi There

I wanted to thank everyone for all of their suggestions regarding
merging images. I think I should have made myself a bit more clear...

We have a Zeiss LSM 510 and are capable of scanning up to 4 different
fluorophores. We have generated several images in which there is a
'green' channel (FITC), 'red' channel (cy3) 'pink' channel (cy5) and
'blue' channel (AMCA/DAPI).

First, when I try to open these 4 color images using the LSM toolbox,
one of the channels is lost. I guess I would have to save each channel
separately to get around this.

(I am quite familiar with photoshop and to avoid problems I save each
channel as a grey scale image and then save the RBG channels in the
appropriate channels. i then create an additional channel to add the
final color; then to save the image, I just merge everything and save as
a jpeg, and for panel shots I can save each channel as a separate
channel).

I discovered the 'RGB to Montage' option within 'color functions' in the
plugins menu. This works great for RGB merged images, but I was
wondering if this can be done with the additional color ... it is simple
enough to create a panel with my LSM software, was just curious if
ImageJ could do something similar/ if there was a way to modify this
macro to include an additional channel/ box in the options that pop up
when creating the panel

Thanks again for the comments,
-Surita




------------------------



Dr. Sharmistha Paul
Department of Materials Science
Queen Mary College, University of London
Mile End Road,London E1, 4NS.
Tel: [phone],
Fax: [phone]



Dr. Sharmistha Paul, Lecturer
Department of Engineering Science
Haldia Institute of Technology
PO - Hatiberia,Haldia,Dist. - Purba Medinipur.
Pin - 721657, West Bengal, India
website: http://www.hit-haldia.net/
Phone [phone] (Ext. 225)
Fax [phone]
 
---------------------------------
Do you Yahoo!?
 Everyone is raving about the  all-new Yahoo! Mail.

[Daniel White]

Surita,

BioImageXD should open your .lsm files from the zeiss lsm 510.
Each colour channel will be listed in the file tree.
THen you can re set the colour of each channel with the colour  
transfer function editor
(click the coloured bar, and choose a new colour)
Then you can select all 4 channels (shift click) in the file tree,
and then click there Merge icon on the top bar.
This will merge the 4 colour channels into a RGB dataset
which can be 3D rendered (if its a stack), saved as an image etc.

www.bioimagexd.org

Dan


On 4 Oct 2006, at 07:00, IMAGEJ automatic digest system wrote:

> Date:    Tue, 3 Oct 2006 06:54:01 -0700
> From:    sharmistha paul <[hidden email]>
> Subject: Re: color merge
>
> Surita Banwait <[hidden email]> wrote:  Hi There
>
> I wanted to thank everyone for all of their suggestions regarding
> merging images. I think I should have made myself a bit more clear...
>
> We have a Zeiss LSM 510 and are capable of scanning up to 4 different
> fluorophores. We have generated several images in which there is a
> 'green' channel (FITC), 'red' channel (cy3) 'pink' channel (cy5) and
> 'blue' channel (AMCA/DAPI).
>
> First, when I try to open these 4 color images using the LSM toolbox,
> one of the channels is lost. I guess I would have to save each channel
> separately to get around this.
>
> (I am quite familiar with photoshop and to avoid problems I save each
> channel as a grey scale image and then save the RBG channels in the
> appropriate channels. i then create an additional channel to add the
> final color; then to save the image, I just merge everything and  
> save as
> a jpeg, and for panel shots I can save each channel as a separate
> channel).
>
> I discovered the 'RGB to Montage' option within 'color functions'  
> in the
> plugins menu. This works great for RGB merged images, but I was
> wondering if this can be done with the additional color ... it is  
> simple
> enough to create a panel with my LSM software, was just curious if
> ImageJ could do something similar/ if there was a way to modify this
> macro to include an additional channel/ box in the options that pop up
> when creating the panel
>
> Thanks again for the comments,
> -Surita

Dr. Daniel James White BSc. (Hons.) PhD
Bioimaging Coordinator
Nanoscience Centre and Department of Biological and Environmental  
Sciences
Division of Molecular Recognition
Ambiotica C242
PO Box 35
University of Jyväskylä
Jyväskylä
FIN 40014
Finland

[phone] (work)
[phone] (mobile)
http://www.bioimagexd.org
http://www.chalkie.org.uk
[hidden email]
[hidden email]