Merge Multiple Images & Cut Through all of Them

[Marina]

Hello All,

I am doing fluorescence microscopy and have four fluorescence channels, each saved as a tiff file.
I wish to merge these files, cut out objects in the same place for each one, then split them again.

When I had only three channels, this was easy: Image > Color > Merge Channels, use the lasso and backspace to edit what I needed and then Image > Color > Split Channels.
However I cannot do this with four channels as Split Channels only splits into red, green and blue despite me merging with red, green, blue and grey.

The solution I have come up with it to form a stack with my images instead of merging and then use the lasso and Edit > Clear to delete the selection in every channel, however this comes up with an "are you sure?" dialogue box and is tedious as I have many cuts and many images to process.

Is there a way for me to split my merged tiffs back into their four channels?
Or does anyone know of another solution to ensure that my cut outs are in the same place for all my channels?

Many thanks,

Marina

[Swayne, Theresa C.]

Hi Marina,

When you merge channels, check the box “Create Composite”.

Then you will have an image with a slider at the bottom where you can select each channel in turn to do the editing.

The composite image preserves the channel information in your original images.

As a side benefit, the ROI you draw with the lasso will be in the same place automatically.

As an alternative, to generate an ROI in the same place in multiple images, you can use the ROI Manager.
(user guide: https://imagej.nih.gov/ij/docs/guide/146-30.html#sub:ROI-Manager…)

After you draw the ROI, press T to add it to the manager.

To apply the ROI to another image, click on the other image window, then click on the ROI name in the ROI Manager.

Hope this helps,
Theresa


On Jun 9, 2016, at 10:58 AM, Marina <[hidden email]<mailto:[hidden email]>> wrote:

When I had only three channels, this was easy: Image > Color > Merge
Channels, use the lasso and backspace to edit what I needed and then Image >
Color > Split Channels.
However I cannot do this with four channels as Split Channels only splits
into red, green and blue despite me merging with red, green, blue and grey.



…

Or does anyone know of another solution to ensure that my cut outs are in
the same place for all my channels?

------------------------------------
Theresa Swayne, Ph.D.
Manager
Confocal and Specialized Microscopy Shared Resource<http://hiccc.columbia.edu/research/sharedresources/confocal>

Herbert Irving Comprehensive Cancer Center
Columbia University Medical Center
1130 St. Nicholas Ave., Room 222A
New York, NY 10032
Phone: [phone]
[hidden email]<mailto:[hidden email]>


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[Glen MacDonald-2]

Dear Marina,
Are you merging your channels into Composite Hyperstacks? Image>Color>Merge Channels, make certain the ‘Create Composite’ box is checked.  All selection operations are possible with composite stacks.  
Alternatively on single channel images, create your selection on 1 channel, open the ROIManager  and click on ‘Add (t)’ .  Now select each channel in turn, click on the ROI to apply it and perform your operation. Finally, merge the channels.

 regards,

Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

> On Jun 9, 2016, at 7:58 AM, Marina <[hidden email]> wrote:
>
> Hello All,
>
> I am doing fluorescence microscopy and have four fluorescence channels, each
> saved as a tiff file.
> I wish to merge these files, cut out objects in the same place for each one,
> then split them again.
>
> When I had only three channels, this was easy: Image > Color > Merge
> Channels, use the lasso and backspace to edit what I needed and then Image >
> Color > Split Channels.
> However I cannot do this with four channels as Split Channels only splits
> into red, green and blue despite me merging with red, green, blue and grey.
>
> The solution I have come up with it to form a stack with my images instead
> of merging and then use the lasso and Edit > Clear to delete the selection
> in every channel, however this comes up with an "are you sure?" dialogue box
> and is tedious as I have many cuts and many images to process.
>
> Is there a way for me to split my merged tiffs back into their four
> channels?
> Or does anyone know of another solution to ensure that my cut outs are in
> the same place for all my channels?
>
> Many thanks,
>
> Marina
>
>
>
> --
> View this message in context: http://imagej.1557.x6.nabble.com/Merge-Multiple-Images-Cut-Through-all-of-Them-tp5016617.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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[Jan Eglinger]

Hi all,

another way to transfer an ROI from one image to another is the
   'Edit > Selection > Restore Selection'
command.

* Activate the image containing the desired selection (by clicking the
title bar or using your OS's mechanism to cycle through windows, e.g.
Alt-Tab)

* Activate the image where you want to transfer the ROI

* Press Shift-E

Done.
Hope that helps,
Jan



On 09.06.16 21:20, Swayne, Theresa C. wrote:

>
> Hi Marina,
>
> When you merge channels, check the box “Create Composite”.
>
> Then you will have an image with a slider at the bottom where you can select each channel in turn to do the editing.
>
> The composite image preserves the channel information in your original images.
>
> As a side benefit, the ROI you draw with the lasso will be in the same place automatically.
>
> As an alternative, to generate an ROI in the same place in multiple images, you can use the ROI Manager.
> (user guide: https://imagej.nih.gov/ij/docs/guide/146-30.html#sub:ROI-Manager…)
>
> After you draw the ROI, press T to add it to the manager.
>
> To apply the ROI to another image, click on the other image window, then click on the ROI name in the ROI Manager.
>
> Hope this helps,
> Theresa
>
>
> On Jun 9, 2016, at 10:58 AM, Marina <[hidden email]<mailto:[hidden email]>> wrote:
>
> When I had only three channels, this was easy: Image > Color > Merge
> Channels, use the lasso and backspace to edit what I needed and then Image >
> Color > Split Channels.
> However I cannot do this with four channels as Split Channels only splits
> into red, green and blue despite me merging with red, green, blue and grey.
>
>
>
> …
>
> Or does anyone know of another solution to ensure that my cut outs are in
> the same place for all my channels?
>
> ------------------------------------
> Theresa Swayne, Ph.D.
> Manager
> Confocal and Specialized Microscopy Shared Resource<http://hiccc.columbia.edu/research/sharedresources/confocal>
>
> Herbert Irving Comprehensive Cancer Center
> Columbia University Medical Center
> 1130 St. Nicholas Ave., Room 222A
> New York, NY 10032
> Phone: [phone]
> [hidden email]<mailto:[hidden email]>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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[Marina]

Hi Theresa,

The reason I have not used composities is that when I create a composite it only has three channels, despite me including four in the "Merge Channels" option. I wish to analyse each channel separately after cutting out the areas I wish to exclude.

Also: when I use the lasso on one channel and hit "delete" it only deletes the area on that channel, as opposed to on all three.

Best wishes,

Marina

Swayne, Theresa C. wrote

When you merge channels, check the box “Create Composite”.

Then you will have an image with a slider at the bottom where you can select each channel in turn to do the editing.

The composite image preserves the channel information in your original images.

[Marina]

Hi all,

The best method I have developed thus far is to work on the stack (as I previously mentioned) but I have now created a macro with an assigned keyboard shortcut for the Edit > Clear component (this avoids the "are you sure?" window so is just one button).

This solution works for me as it allows me to quickly cut out sections from all the channels in the same place (I have many cuts to do per image and many images), and then allows me to re-split the channels to the four (not cut) images and analyse them.

However I am still unsatisfied by the inability to split a merged image with more than three channels and by my composite also only having three channels despite me selecting four.
Does anyone have any solutions or insight for this?

Best wishes,

Marina

Marina wrote

The solution I have come up with it to form a stack with my images instead of merging and then use the lasso and Edit > Clear to delete the selection in every channel, however this comes up with an "are you sure?" dialogue box and is tedious as I have many cuts and many images to process.

[Swayne, Theresa C.]

Hi Marina,

You can merge 4 channels if they are each single-color images in “grayscale” format (says "8-bit" or "16-bit" at the top of the image window, NOT “RGB").

It could be that your images are already RGB format before merging.

If you convert them to 8-bit or 16-bit before merging, do you still have the problem?

If that doesn’t solve it, could you post some sample images on imgur or on the forum (forum.imagej.net<http://forum.imagej.net>)?

Clearing areas is channel-specific in composite images, on purpose. You will have to scroll through the channels and press delete on each one. The advantage is that your lasso selection stays in the same place automatically.

Hope this helps.

Best,
Theresa

On Jun 14, 2016, at 11:47 AM, Marina <[hidden email]<mailto:[hidden email]>> wrote:

The reason I have not used composities is that when I create a composite it
only has three channels, despite me including four in the "Merge Channels"
option. I wish to analyse each channel separately after cutting out the
areas I wish to exclude.

Also: when I use the lasso on one channel and hit "delete" it only deletes
the area on that channel, as opposed to on all three.

Best wishes,

Marina



Swayne, Theresa C. wrote
When you merge channels, check the box “Create Composite”.

Then you will have an image with a slider at the bottom where you can
select each channel in turn to do the editing.

The composite image preserves the channel information in your original
images.





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View this message in context: http://imagej.1557.x6.nabble.com/Merge-Multiple-Images-Cut-Through-all-of-Them-tp5016617p5016638.html
Sent from the ImageJ mailing list archive at Nabble.com<http://nabble.com>.

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------------------------------------
Theresa Swayne, Ph.D.
Manager
Confocal and Specialized Microscopy Shared Resource<http://hiccc.columbia.edu/research/sharedresources/confocal>

Herbert Irving Comprehensive Cancer Center
Columbia University Medical Center
1130 St. Nicholas Ave., Room 222A
New York, NY 10032
Phone: [phone]
[hidden email]<mailto:[hidden email]>


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