ImageJ

non-destructive 2 channel merge question

_‹ Previous Topic Next Topic _›

Classic

List

Threaded

8 messages Options

Mike A

non-destructive 2 channel merge question

Hi folks,

I am using imageJ v1.44n6 and I am working with a 2 channel (red/green) timelapse movie with Z-sections.

I open this file with LOCI bioformats and use the “split channels” function. I then use the following to get a red/green overlay which i then save to .tif: image > color > merge channels > select the 2 channels > leave “create composite” blank > click ok. However, I noticed that if prior to merging the 2 channels I have adjusted pixel intensities using the window/level tool, these are destructively carried over into my merged RGB file. Is there a way to avoid this?

I want to create an image where red and green are overlayed, but where the pixel intensities in both the red and green channels represent those of my raw data. Really, the question is how does one create a red green overlay and save to .tif without destructively altering pixel intensity values. Is it just a matter of avoiding using the window/level tool before merging?? I like to be as non-destructive as possible before getting my output .tif file into photoshop.

Many thanks for your help.

Mike

Glen MacDonald-2

Re: non-destructive 2 channel merge question

Dear Mike,
that is the nature of RGB conversion, the default assumption is you have selected the interesting portion of the source histogram. Use Adjust>B&C or >Color Balance on the Composite source images to either Set channels to 0-bit depth (eg 0 to 4095) and apply to all channels, or check the histograms and set to min/max for each channel. Then Image>Color>merge channels to RGB. alternatively, you can make your adjustments on merged Composite images then Image>Type>RGB.

Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

On Mar 17, 2011, at 6:39 AM, trshrthrth rdthjdrtjdrtj wrote:

> Hi folks,
>
> I am using imageJ v1.44n6 and I am working with a 2 channel (red/green) timelapse movie with Z-sections.
>
> I open this file with LOCI bioformats and use the “split channels” function. I then use the following to get a red/green overlay which i then save to .tif: image > color > merge channels > select the 2 channels > leave “create composite” blank > click ok. However, I noticed that if prior to merging the 2 channels I have adjusted pixel intensities using the window/level tool, these are destructively carried over into my merged RGB file. Is there a way to avoid this?
>
> I want to create an image where red and green are overlayed, but where the pixel intensities in both the red and green channels represent those of my raw data. Really, the question is how does one create a red green overlay and save to .tif without destructively altering pixel intensity values. Is it just a matter of avoiding using the window/level tool before merging?? I like to be as non-destructive as possible before getting my output .tif file into photoshop.
>
> Many thanks for your help.
>
> Mike

ctrueden

Re: non-destructive 2 channel merge question

In reply to this post by Mike A

Hi Mike,

The "Merge channels" command will destructively remap your data to create an
RGB image. The problem with RGB images is that they are limited to exactly
three 8-bit color channels.

I suggest you avoid using RGB images, and instead use the "composite image"
feature. You can display multi-channel 16-bit data, for example, with a red
and a green channel, without altering the color values.

You can save a composite image to a TIFF with each channel as a separate
plane, preserving the bit depth.

You can also use the Bio-Formats importer to display your image as red-green
without all the steps you describe. Just change the "Color mode" to
"Composite" and it should do what you want.

-Curtis

On Thu, Mar 17, 2011 at 8:39 AM, trshrthrth rdthjdrtjdrtj <
[hidden email]> wrote:

> Hi folks,
>
> I am using imageJ v1.44n6 and I am working with a 2 channel (red/green)
> timelapse movie with Z-sections.
>
> I open this file with LOCI bioformats and use the “split channels”
> function. I then use the following to get a red/green overlay which i then
> save to .tif: image > color > merge channels > select the 2 channels > leave
> “create composite” blank > click ok. However, I noticed that if prior to
> merging the 2 channels I have adjusted pixel intensities using the
> window/level tool, these are destructively carried over into my merged RGB
> file. Is there a way to avoid this?
>
> I want to create an image where red and green are overlayed, but where the
> pixel intensities in both the red and green channels represent those of my
> raw data. Really, the question is how does one create a red green overlay
> and save to .tif without destructively altering pixel intensity values. Is
> it just a matter of avoiding using the window/level tool before merging?? I
> like to be as non-destructive as possible before getting my output .tif file
> into photoshop.
>
> Many thanks for your help.
>
> Mike
>

Michael P Ellis

Re: non-destructive 2 channel merge question

In reply to this post by Mike A

Mike, hi

As already suggested, you are probably best off getting your 2 channel, time lapse image in the form of a hyperstack and using the composite display mode to display the merged the red and green channels.

I have written a plugin called "Composite Adjuster" that lets you conveniently adjust levels and colours whilst such a hyperstack is being animated. I am not sure if that would be of any interest to you but details here: http://www.dsuk.biz/DSUK/SmartCaptureLite.html

A YouTube clip of it in action here: http://www.youtube.com/embed/g0hoRLxBlWE?rel=0&hd=1

Regards -- Michael Ellis

On 17 Mar 2011, at 13:39, trshrthrth rdthjdrtjdrtj wrote:

Mike A

Re: non-destructive 2 channel merge question

In reply to this post by ctrueden

Thank you Glen/Curtis for your helpful comments. Curtis: thanks that “color mode” to “composite” is useful to know.

The trouble is, I would love to stick with composite images, but the situation is a little more complex as i need to us the reslice command on both red and green channels separately... and then generate a red/green overlay of these two channels together. The reslice command cannot be used with 2-channel composite images?

I think i should describe exactly what i am trying to do, i wanted to avoid this to keep things simple but it might be neccessary.

I open my 2 channel (red/green) timelapse movie with Z-sections using LOCI and imageJ v1.44n6 and maxintensity project all my Z’s. I then mark up regions of interest using the line tool that im interested in and add each line to the ROI manager. I then split my channels with LOCI and run a macro on each channel (macro kindly written by Wayne - attached at the bottom of this message). This macro takes each line selection in the ROI Manager, re-slices using a slice_count of 7, rotates the reslice, does a Max Intensity projection and then saves the projections in TIFF format. Essentially the result is a bunch of reslices saved as tif files (reslices from each channel are saved to separate folders).

However, i also need to show the individual reslices of each channel together in colour (i prefer the individual channel reslices to be greyscale). The trouble is, how to combine in the least destructive way a reslice of the two channels in which one channel coloured red is overlayed on the other channel coloured green. Its important that all three reslices look like they have had the same level adjustments. Not sure how to do this ! should i just merge the output reslices for each channel and thats that?

Thanks for your help.

Mike

++macro that im using:

setBatchMode(true);
dir = getDirectory("Choose a Directory");
n = roiManager("count");
for (i=0; i<n; i++) {
roiManager("Select", i);
run("Reslice [/]...", "slice_count=7 rotate");
id = getImageID;
run("Z Project...", "projection=[Max Intensity]");
saveAs("Tiff", dir+i+".tif");
close();
selectImage(id);
close();
}
From: Curtis Rueden
Sent: Thursday, March 17, 2011 6:16 PM
To: ImageJ Interest Group
Cc: trshrthrth rdthjdrtjdrtj
Subject: Re: non-destructive 2 channel merge question

Hi Mike,

The "Merge channels" command will destructively remap your data to create an RGB image. The problem with RGB images is that they are limited to exactly three 8-bit color channels.

I suggest you avoid using RGB images, and instead use the "composite image" feature. You can display multi-channel 16-bit data, for example, with a red and a green channel, without altering the color values.

You can save a composite image to a TIFF with each channel as a separate plane, preserving the bit depth.

You can also use the Bio-Formats importer to display your image as red-green without all the steps you describe. Just change the "Color mode" to "Composite" and it should do what you want.

-Curtis

On Thu, Mar 17, 2011 at 8:39 AM, trshrthrth rdthjdrtjdrtj <[hidden email]> wrote:

Hi folks,

I am using imageJ v1.44n6 and I am working with a 2 channel (red/green) timelapse movie with Z-sections.

I open this file with LOCI bioformats and use the “split channels” function. I then use the following to get a red/green overlay which i then save to .tif: image > color > merge channels > select the 2 channels > leave “create composite” blank > click ok. However, I noticed that if prior to merging the 2 channels I have adjusted pixel intensities using the window/level tool, these are destructively carried over into my merged RGB file. Is there a way to avoid this?

I want to create an image where red and green are overlayed, but where the pixel intensities in both the red and green channels represent those of my raw data. Really, the question is how does one create a red green overlay and save to .tif without destructively altering pixel intensity values. Is it just a matter of avoiding using the window/level tool before merging?? I like to be as non-destructive as possible before getting my output .tif file into photoshop.

Many thanks for your help.

Mike

Glen MacDonald-2

Re: non-destructive 2 channel merge question

On Mar 17, 2011, at 11:47 AM, trshrthrth rdthjdrtjdrtj wrote:

> Thank you Glen/Curtis for your helpful comments. Curtis: thanks that “color mode” to “composite” is useful to know.
>
> The trouble is, I would love to stick with composite images, but the situation is a little more complex as i need to us the reslice command on both red and green channels separately... and then generate a red/green overlay of these two channels together. The reslice command cannot be used with 2-channel composite images?
I just opened 1 channel and 2 channel images, that had been digitized to 12 bits and saved as 16-bit. Imported with LOCI/Composite, ImageJ 1.45b4, Mac OS 10.6.6, Java 1.6.0_24 64-bit
Reslice and Z Project both provide 16-bit composite results.

Ran your macro on a merged composite image and also obtained 16-bit results. Note that depending on your source, LOCI Composite may make both channels the same color. In that case either select 'Custom' color in LOCI or select the second channel in the composite window and use Image>color>Channels to set the correct color.

A key question is what constitutes "look like they have had the same level adjustments." Are you wanting to illustrate relative staining intensities, histogram stretch for maximum signals, lack of weak intensities, prevent the weakest channel from being overwhelmed in the merged result....? RGB (required for display by QuickTime, Media Player, Powerpoint, Keynote, etc) is inherently destructive. You can only direct the damage to selected parts of the histogram. the Autoscale in the LOCI plugin will scale the histogram to the min/max of the image, without it the histogram is scaled to the digitization bit-depth (if known).

Glen

>
> I think i should describe exactly what i am trying to do, i wanted to avoid this to keep things simple but it might be neccessary.
>
> I open my 2 channel (red/green) timelapse movie with Z-sections using LOCI and imageJ v1.44n6 and maxintensity project all my Z’s. I then mark up regions of interest using the line tool that im interested in and add each line to the ROI manager. I then split my channels with LOCI and run a macro on each channel (macro kindly written by Wayne - attached at the bottom of this message). This macro takes each line selection in the ROI Manager, re-slices using a slice_count of 7, rotates the reslice, does a Max Intensity projection and then saves the projections in TIFF format. Essentially the result is a bunch of reslices saved as tif files (reslices from each channel are saved to separate folders).
>
> However, i also need to show the individual reslices of each channel together in colour (i prefer the individual channel reslices to be greyscale). The trouble is, how to combine in the least destructive way a reslice of the two channels in which one channel coloured red is overlayed on the other channel coloured green. Its important that all three reslices look like they have had the same level adjustments. Not sure how to do this ! should i just merge the output reslices for each channel and thats that?
>
> Thanks for your help.
>
> Mike
>
> ++macro that im using:
>
> setBatchMode(true);
> dir = getDirectory("Choose a Directory");
> n = roiManager("count");
> for (i=0; i<n; i++) {
> roiManager("Select", i);
> run("Reslice [/]...", "slice_count=7 rotate");
> id = getImageID;
> run("Z Project...", "projection=[Max Intensity]");
> saveAs("Tiff", dir+i+".tif");
> close();
> selectImage(id);
> close();
> }
> From: Curtis Rueden
> Sent: Thursday, March 17, 2011 6:16 PM
> To: ImageJ Interest Group
> Cc: trshrthrth rdthjdrtjdrtj
> Subject: Re: non-destructive 2 channel merge question
>
> Hi Mike,
>
> The "Merge channels" command will destructively remap your data to create an RGB image. The problem with RGB images is that they are limited to exactly three 8-bit color channels.
>
> I suggest you avoid using RGB images, and instead use the "composite image" feature. You can display multi-channel 16-bit data, for example, with a red and a green channel, without altering the color values.
>
> You can save a composite image to a TIFF with each channel as a separate plane, preserving the bit depth.
>
> You can also use the Bio-Formats importer to display your image as red-green without all the steps you describe. Just change the "Color mode" to "Composite" and it should do what you want.
>
> -Curtis
>
>
> On Thu, Mar 17, 2011 at 8:39 AM, trshrthrth rdthjdrtjdrtj <[hidden email]> wrote:
>
> Hi folks,
>
> I am using imageJ v1.44n6 and I am working with a 2 channel (red/green) timelapse movie with Z-sections.
>
> I open this file with LOCI bioformats and use the “split channels” function. I then use the following to get a red/green overlay which i then save to .tif: image > color > merge channels > select the 2 channels > leave “create composite” blank > click ok. However, I noticed that if prior to merging the 2 channels I have adjusted pixel intensities using the window/level tool, these are destructively carried over into my merged RGB file. Is there a way to avoid this?
>
> I want to create an image where red and green are overlayed, but where the pixel intensities in both the red and green channels represent those of my raw data. Really, the question is how does one create a red green overlay and save to .tif without destructively altering pixel intensity values. Is it just a matter of avoiding using the window/level tool before merging?? I like to be as non-destructive as possible before getting my output .tif file into photoshop.
>
> Many thanks for your help.
>
> Mike

Mike A

Re: non-destructive 2 channel merge question

Thanks Glen for checking that out. I think the problem is this. Thanks to
the mailing lists advice its clear i need to reslice a composite stack to
prevent destruction of pixel data. However, i have a 2 channel timelapse
movie with z-sections. If i open this with LOCI with colour mode set to
composite and then project my z-sections im left with a composite stack with
2 channels and lots of timepoints. What i am trying to do is reslice
through time (to generate a kymograph) rather than through the Z-sections
which are now all projected. When I try to reslice my 2channel composite
stack with timepoints i get the error "cannot reslice z=1 hyperstacks".
Presumably the reslice command isnt happy reslicing through time in
composite stacks?? Is there a way around this?

Mike

-----Original Message-----
From: Glen MacDonald
Sent: Thursday, March 17, 2011 7:55 PM
To: [hidden email]
Subject: Re: non-destructive 2 channel merge question

On Mar 17, 2011, at 11:47 AM, trshrthrth rdthjdrtjdrtj wrote:

> Thank you Glen/Curtis for your helpful comments. Curtis: thanks that
> “color mode” to “composite” is useful to know.
>
> The trouble is, I would love to stick with composite images, but the
> situation is a little more complex as i need to us the reslice command on
> both red and green channels separately... and then generate a red/green
> overlay of these two channels together. The reslice command cannot be
> used with 2-channel composite images?
I just opened 1 channel and 2 channel images, that had been digitized to 12
bits and saved as 16-bit. Imported with LOCI/Composite, ImageJ 1.45b4, Mac
OS 10.6.6, Java 1.6.0_24 64-bit
Reslice and Z Project both provide 16-bit composite results.

Ran your macro on a merged composite image and also obtained 16-bit results.
Note that depending on your source, LOCI Composite may make both channels
the same color. In that case either select 'Custom' color in LOCI or select
the second channel in the composite window and use Image>color>Channels to
set the correct color.

A key question is what constitutes "look like they have had the same level
adjustments." Are you wanting to illustrate relative staining intensities,
histogram stretch for maximum signals, lack of weak intensities, prevent the
weakest channel from being overwhelmed in the merged result....? RGB
(required for display by QuickTime, Media Player, Powerpoint, Keynote, etc)
is inherently destructive. You can only direct the damage to selected parts
of the histogram. the Autoscale in the LOCI plugin will scale the
histogram to the min/max of the image, without it the histogram is scaled to
the digitization bit-depth (if known).

Glen

>
> I think i should describe exactly what i am trying to do, i wanted to
> avoid this to keep things simple but it might be neccessary.
>
> I open my 2 channel (red/green) timelapse movie with Z-sections using LOCI
> and imageJ v1.44n6 and maxintensity project all my Z’s. I then mark up
> regions of interest using the line tool that im interested in and add each
> line to the ROI manager. I then split my channels with LOCI and run a
> macro on each channel (macro kindly written by Wayne - attached at the
> bottom of this message). This macro takes each line selection in the ROI
> Manager, re-slices using a slice_count of 7, rotates the reslice, does a
> Max Intensity projection and then saves the projections in TIFF format.
> Essentially the result is a bunch of reslices saved as tif files (reslices
> from each channel are saved to separate folders).
>
> However, i also need to show the individual reslices of each channel
> together in colour (i prefer the individual channel reslices to be
> greyscale). The trouble is, how to combine in the least destructive way a
> reslice of the two channels in which one channel coloured red is overlayed
> on the other channel coloured green. Its important that all three
> reslices look like they have had the same level adjustments. Not sure how
> to do this ! should i just merge the output reslices for each channel and
> thats that?
>
> Thanks for your help.
>
> Mike
>
> ++macro that im using:
>
> setBatchMode(true);
> dir = getDirectory("Choose a Directory");
> n = roiManager("count");
> for (i=0; i<n; i++) {
> roiManager("Select", i);
> run("Reslice [/]...", "slice_count=7 rotate");
> id = getImageID;
> run("Z Project...", "projection=[Max Intensity]");
> saveAs("Tiff", dir+i+".tif");
> close();
> selectImage(id);
> close();
> }
> From: Curtis Rueden
> Sent: Thursday, March 17, 2011 6:16 PM
> To: ImageJ Interest Group
> Cc: trshrthrth rdthjdrtjdrtj
> Subject: Re: non-destructive 2 channel merge question
>
> Hi Mike,
>
> The "Merge channels" command will destructively remap your data to create
> an RGB image. The problem with RGB images is that they are limited to
> exactly three 8-bit color channels.
>
> I suggest you avoid using RGB images, and instead use the "composite
> image" feature. You can display multi-channel 16-bit data, for example,
> with a red and a green channel, without altering the color values.
>
> You can save a composite image to a TIFF with each channel as a separate
> plane, preserving the bit depth.
>
> You can also use the Bio-Formats importer to display your image as
> red-green without all the steps you describe. Just change the "Color mode"
> to "Composite" and it should do what you want.
>
> -Curtis
>
>
> On Thu, Mar 17, 2011 at 8:39 AM, trshrthrth rdthjdrtjdrtj
> <[hidden email]> wrote:
>
> Hi folks,
>
> I am using imageJ v1.44n6 and I am working with a 2 channel (red/green)
> timelapse movie with Z-sections.
>
> I open this file with LOCI bioformats and use the “split channels”
> function. I then use the following to get a red/green overlay which i then
> save to .tif: image > color > merge channels > select the 2 channels >
> leave “create composite” blank > click ok. However, I noticed that if
> prior to merging the 2 channels I have adjusted pixel intensities using
> the window/level tool, these are destructively carried over into my merged
> RGB file. Is there a way to avoid this?
>
> I want to create an image where red and green are overlayed, but where
> the pixel intensities in both the red and green channels represent those
> of my raw data. Really, the question is how does one create a red green
> overlay and save to .tif without destructively altering pixel intensity
> values. Is it just a matter of avoiding using the window/level tool
> before merging?? I like to be as non-destructive as possible before
> getting my output .tif file into photoshop.
>
> Many thanks for your help.
>
> Mike

Glen MacDonald-2

Re: non-destructive 2 channel merge question

Can you put a small dataset somewhere I can download it? Would it help to reslice your z-stacks in the x or y axis (e.g. left or top) then use Z-Project to create the kymograph views. I don't have a 4D dataset handy to see if that will work without splitting your timeseries into stacks (LOCI can do this) then merge the projections into a movie.

Glen
On Mar 18, 2011, at 3:09 AM, trshrthrth rdthjdrtjdrtj wrote:

> Thanks Glen for checking that out. I think the problem is this. Thanks to the mailing lists advice its clear i need to reslice a composite stack to prevent destruction of pixel data. However, i have a 2 channel timelapse movie with z-sections. If i open this with LOCI with colour mode set to composite and then project my z-sections im left with a composite stack with 2 channels and lots of timepoints. What i am trying to do is reslice through time (to generate a kymograph) rather than through the Z-sections which are now all projected. When I try to reslice my 2channel composite stack with timepoints i get the error "cannot reslice z=1 hyperstacks". Presumably the reslice command isnt happy reslicing through time in composite stacks?? Is there a way around this?
>
> Mike
>
> -----Original Message----- From: Glen MacDonald
> Sent: Thursday, March 17, 2011 7:55 PM
> To: [hidden email]
> Subject: Re: non-destructive 2 channel merge question
>
> On Mar 17, 2011, at 11:47 AM, trshrthrth rdthjdrtjdrtj wrote:
>
>> Thank you Glen/Curtis for your helpful comments. Curtis: thanks that “color mode” to “composite” is useful to know.
>>
>> The trouble is, I would love to stick with composite images, but the situation is a little more complex as i need to us the reslice command on both red and green channels separately... and then generate a red/green overlay of these two channels together. The reslice command cannot be used with 2-channel composite images?
> I just opened 1 channel and 2 channel images, that had been digitized to 12 bits and saved as 16-bit. Imported with LOCI/Composite, ImageJ 1.45b4, Mac OS 10.6.6, Java 1.6.0_24 64-bit
> Reslice and Z Project both provide 16-bit composite results.
>
> Ran your macro on a merged composite image and also obtained 16-bit results. Note that depending on your source, LOCI Composite may make both channels the same color. In that case either select 'Custom' color in LOCI or select the second channel in the composite window and use Image>color>Channels to set the correct color.
>
> A key question is what constitutes "look like they have had the same level adjustments." Are you wanting to illustrate relative staining intensities, histogram stretch for maximum signals, lack of weak intensities, prevent the weakest channel from being overwhelmed in the merged result....? RGB (required for display by QuickTime, Media Player, Powerpoint, Keynote, etc) is inherently destructive. You can only direct the damage to selected parts of the histogram. the Autoscale in the LOCI plugin will scale the histogram to the min/max of the image, without it the histogram is scaled to the digitization bit-depth (if known).
>
> Glen
>>
>> I think i should describe exactly what i am trying to do, i wanted to avoid this to keep things simple but it might be neccessary.
>>
>> I open my 2 channel (red/green) timelapse movie with Z-sections using LOCI and imageJ v1.44n6 and maxintensity project all my Z’s. I then mark up regions of interest using the line tool that im interested in and add each line to the ROI manager. I then split my channels with LOCI and run a macro on each channel (macro kindly written by Wayne - attached at the bottom of this message). This macro takes each line selection in the ROI Manager, re-slices using a slice_count of 7, rotates the reslice, does a Max Intensity projection and then saves the projections in TIFF format. Essentially the result is a bunch of reslices saved as tif files (reslices from each channel are saved to separate folders).
>>
>> However, i also need to show the individual reslices of each channel together in colour (i prefer the individual channel reslices to be greyscale). The trouble is, how to combine in the least destructive way a reslice of the two channels in which one channel coloured red is overlayed on the other channel coloured green. Its important that all three reslices look like they have had the same level adjustments. Not sure how to do this ! should i just merge the output reslices for each channel and thats that?
>>
>> Thanks for your help.
>>
>> Mike
>>
>> ++macro that im using:
>>
>> setBatchMode(true);
>> dir = getDirectory("Choose a Directory");
>> n = roiManager("count");
>> for (i=0; i<n; i++) {
>> roiManager("Select", i);
>> run("Reslice [/]...", "slice_count=7 rotate");
>> id = getImageID;
>> run("Z Project...", "projection=[Max Intensity]");
>> saveAs("Tiff", dir+i+".tif");
>> close();
>> selectImage(id);
>> close();
>> }
>> From: Curtis Rueden
>> Sent: Thursday, March 17, 2011 6:16 PM
>> To: ImageJ Interest Group
>> Cc: trshrthrth rdthjdrtjdrtj
>> Subject: Re: non-destructive 2 channel merge question
>>
>> Hi Mike,
>>
>> The "Merge channels" command will destructively remap your data to create an RGB image. The problem with RGB images is that they are limited to exactly three 8-bit color channels.
>>
>> I suggest you avoid using RGB images, and instead use the "composite image" feature. You can display multi-channel 16-bit data, for example, with a red and a green channel, without altering the color values.
>>
>> You can save a composite image to a TIFF with each channel as a separate plane, preserving the bit depth.
>>
>> You can also use the Bio-Formats importer to display your image as red-green without all the steps you describe. Just change the "Color mode" to "Composite" and it should do what you want.
>>
>> -Curtis
>>
>>
>> On Thu, Mar 17, 2011 at 8:39 AM, trshrthrth rdthjdrtjdrtj <[hidden email]> wrote:
>>
>> Hi folks,
>>
>> I am using imageJ v1.44n6 and I am working with a 2 channel (red/green) timelapse movie with Z-sections.
>>
>> I open this file with LOCI bioformats and use the “split channels” function. I then use the following to get a red/green overlay which i then save to .tif: image > color > merge channels > select the 2 channels > leave “create composite” blank > click ok. However, I noticed that if prior to merging the 2 channels I have adjusted pixel intensities using the window/level tool, these are destructively carried over into my merged RGB file. Is there a way to avoid this?
>>
>> I want to create an image where red and green are overlayed, but where the pixel intensities in both the red and green channels represent those of my raw data. Really, the question is how does one create a red green overlay and save to .tif without destructively altering pixel intensity values. Is it just a matter of avoiding using the window/level tool before merging?? I like to be as non-destructive as possible before getting my output .tif file into photoshop.
>>
>> Many thanks for your help.
>>
>> Mike