Help wanted with time-lapse analysis - redirect to using stacks

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Help wanted with time-lapse analysis - redirect to using stacks

Jacqueline Ross
Hi everyone,

I'm hoping that someone has done this before and has a solution in place that I can use or adapt.

I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.

However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.

The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.

I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  +64 9 923 7438

Website:    http://www.auckland.ac.nz/biru




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Re: Help wanted with time-lapse analysis - redirect to using stacks

Straatman, Kees (Dr.)
Dear Jacqui,

One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Advanced Imaging Facility
Centre for Core Biotechnology Services
University of Leicester
www.le.ac.uk/advanced-imaging-facility




-----Original Message-----
From: Jacqueline Ross [mailto:[hidden email]]
Sent: 15 May 2018 05:43
To: [hidden email]
Subject: Help wanted with time-lapse analysis - redirect to using stacks

Hi everyone,

I'm hoping that someone has done this before and has a solution in place that I can use or adapt.

I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.

However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.

The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.

I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  +64 9 923 7438

Website:    http://www.auckland.ac.nz/biru




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Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqueline Ross
Dear Kees,

Thank you very much for that suggestion. I didn't know about that option. Only problem now is that I would like one result (summary) per slice rather than the individual values.

I guess I could split the stack into individual images, combine the ROIs and then measure again.

Kind regards,

Jacqui

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Straatman, Kees (Dr.)
Sent: Tuesday, 15 May 2018 7:16 p.m.
To: [hidden email]
Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks

Dear Jacqui,

One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Advanced Imaging Facility
Centre for Core Biotechnology Services
University of Leicester
www.le.ac.uk/advanced-imaging-facility




-----Original Message-----
From: Jacqueline Ross [mailto:[hidden email]]
Sent: 15 May 2018 05:43
To: [hidden email]
Subject: Help wanted with time-lapse analysis - redirect to using stacks

Hi everyone,

I'm hoping that someone has done this before and has a solution in place that I can use or adapt.

I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.

However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.

The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.

I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.

Kind regards,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  +64 9 923 7438

Website:    http://www.auckland.ac.nz/biru




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Re: Help wanted with time-lapse analysis - redirect to using stacks

G. Esteban Fernandez
Jacqui,

Your approach works for me with the "Confocal Series" sample image but
only if I check the "limit to threshold" box in Set Measurements and
leave the red Threshold overlay activated on the binary stack.  The
macro does scroll through the series.

I wonder if with your dataset you have to use "Stack.setFrame(n)" in
your macro instead of "setSlice(n)", since it is a timelapse not a
z-stack.

-Esteban

On Tue, May 15, 2018 at 3:18 PM, Jacqueline Ross
<[hidden email]> wrote:

> Dear Kees,
>
> Thank you very much for that suggestion. I didn't know about that option. Only problem now is that I would like one result (summary) per slice rather than the individual values.
>
> I guess I could split the stack into individual images, combine the ROIs and then measure again.
>
> Kind regards,
>
> Jacqui
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Straatman, Kees (Dr.)
> Sent: Tuesday, 15 May 2018 7:16 p.m.
> To: [hidden email]
> Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks
>
> Dear Jacqui,
>
> One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.
>
> Best wishes
>
> Kees
>
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Advanced Imaging Facility
> Centre for Core Biotechnology Services
> University of Leicester
> www.le.ac.uk/advanced-imaging-facility
>
>
>
>
> -----Original Message-----
> From: Jacqueline Ross [mailto:[hidden email]]
> Sent: 15 May 2018 05:43
> To: [hidden email]
> Subject: Help wanted with time-lapse analysis - redirect to using stacks
>
> Hi everyone,
>
> I'm hoping that someone has done this before and has a solution in place that I can use or adapt.
>
> I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.
>
> However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.
>
> The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.
>
> I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU)
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI:  +64 9 923 7438
>
> Website:    http://www.auckland.ac.nz/biru
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqueline Ross
Hi Esteban,

Thanks for your reply. I tried your suggestion but can't seem to get it to work. The thing that troubles me is that I am able to get results using the Measure Stack macro but they don't match the results I get when I do the analysis manually.

Kind regards,

Jacqui

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez
Sent: Wednesday, 16 May 2018 10:44 a.m.
To: [hidden email]
Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqui,

Your approach works for me with the "Confocal Series" sample image but only if I check the "limit to threshold" box in Set Measurements and leave the red Threshold overlay activated on the binary stack.  The macro does scroll through the series.

I wonder if with your dataset you have to use "Stack.setFrame(n)" in your macro instead of "setSlice(n)", since it is a timelapse not a z-stack.

-Esteban

On Tue, May 15, 2018 at 3:18 PM, Jacqueline Ross <[hidden email]> wrote:

> Dear Kees,
>
> Thank you very much for that suggestion. I didn't know about that option. Only problem now is that I would like one result (summary) per slice rather than the individual values.
>
> I guess I could split the stack into individual images, combine the ROIs and then measure again.
>
> Kind regards,
>
> Jacqui
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Straatman, Kees (Dr.)
> Sent: Tuesday, 15 May 2018 7:16 p.m.
> To: [hidden email]
> Subject: Re: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Dear Jacqui,
>
> One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.
>
> Best wishes
>
> Kees
>
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Advanced Imaging Facility
> Centre for Core Biotechnology Services University of Leicester
> www.le.ac.uk/advanced-imaging-facility
>
>
>
>
> -----Original Message-----
> From: Jacqueline Ross [mailto:[hidden email]]
> Sent: 15 May 2018 05:43
> To: [hidden email]
> Subject: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Hi everyone,
>
> I'm hoping that someone has done this before and has a solution in place that I can use or adapt.
>
> I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.
>
> However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.
>
> The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.
>
> I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU) School of Medical Sciences
> Faculty of Medical & Health Sciences The University of Auckland
> Private Bag 92019 Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI:  +64 9 923 7438
>
> Website:    http://www.auckland.ac.nz/biru
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqueline Ross
Hi All,

So, those images didn't make it so here is my message again without the two images.

Kind regards,

Jacqui

-----Original Message-----
From: Jacqueline Ross
Sent: Friday, 18 May 2018 6:36 p.m.
To: '[hidden email]' <[hidden email]>
Subject: RE: Help wanted with time-lapse analysis - redirect to using stacks

Hi Kees and Esteban (and anyone else trying to solve this!),

I've now tried using a macro written by Jeffrey Murphy. It relies on having two folders of images. So, I've put the binary mask images in Folder A and the raw images for the other channel in Folder B.

I still get different results when using the macro compared to manual  measurements. It must be something simple but I can't work it out.

Here is my macro so far;

// Jeffrey N. Murphy
// @MurphysLab
// 2015-06-30

// This presumes that the images in Folder A and in Folder B are in the same order!

// Get a list of images in Folder A
image_directory_A = getDirectory("Choose Folder A"); file_list_A = getFileList(image_directory_A); image_list_A = newArray(0); image_count_A = 0; for(i=0; i<file_list_A.length; i++){
        if(endsWith(toLowerCase(file_list_A[i]),"tif")||endsWith(toLowerCase(file_list_A[i]),"png")){
                image_count_A++;
                image_list_A = Array.concat(image_list_A,file_list_A[i]);
        }
}

// Get a list of images in Folder B
image_directory_B = getDirectory("Choose Folder B"); file_list_B = getFileList(image_directory_B); image_list_B = newArray(0); image_count_B = 0; for(i=0; i<file_list_B.length; i++){
        if(endsWith(toLowerCase(file_list_B[i]),"tif")||endsWith(toLowerCase(file_list_B[i]),"png")){
                image_count_B++;
                image_list_B = Array.concat(image_list_B,file_list_B[i]);
        }
}

if(image_count_B == image_count_A){
        for(img_i=0; img_i<image_count_A; img_i++){
                open(image_directory_A + image_list_A[img_i]);
                image_A = getImageID();
                open(image_directory_B + image_list_B[img_i]);
                image_B = getImageID();
               
                // IMAGE A
                selectImage(image_A);
                // do what you need to make your ROI
                                //run("Threshold...");
setAutoThreshold("Moments dark");
run("Create Selection");
roiManager("Add");
                close();
               
                // IMAGE B
                selectImage(image_B);
                // measure what you need using the ROI roiManager("Show All");

                //run("Threshold...");
setAutoThreshold("Moments dark");
run("Set Measurements...", "area mean standard modal min integrated median skewness kurtosis area_fraction limit display redirect=None decimal=4"); roiManager("Select", 0); roiManager("Measure"); roiManager("Delete");
                close();
        }
}
               
Kind regards,

Jacqui
-----Original Message-----
From: Jacqueline Ross
Sent: Friday, 18 May 2018 6:31 p.m.
To: [hidden email]
Subject: RE: Help wanted with time-lapse analysis - redirect to using stacks

Hi Esteban,

Thanks for your reply. I tried your suggestion but can't seem to get it to work. The thing that troubles me is that I am able to get results using the Measure Stack macro but they don't match the results I get when I do the analysis manually.

Kind regards,

Jacqui

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez
Sent: Wednesday, 16 May 2018 10:44 a.m.
To: [hidden email]
Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqui,

Your approach works for me with the "Confocal Series" sample image but only if I check the "limit to threshold" box in Set Measurements and leave the red Threshold overlay activated on the binary stack.  The macro does scroll through the series.

I wonder if with your dataset you have to use "Stack.setFrame(n)" in your macro instead of "setSlice(n)", since it is a timelapse not a z-stack.

-Esteban

On Tue, May 15, 2018 at 3:18 PM, Jacqueline Ross <[hidden email]> wrote:

> Dear Kees,
>
> Thank you very much for that suggestion. I didn't know about that option. Only problem now is that I would like one result (summary) per slice rather than the individual values.
>
> I guess I could split the stack into individual images, combine the ROIs and then measure again.
>
> Kind regards,
>
> Jacqui
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Straatman, Kees (Dr.)
> Sent: Tuesday, 15 May 2018 7:16 p.m.
> To: [hidden email]
> Subject: Re: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Dear Jacqui,
>
> One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.
>
> Best wishes
>
> Kees
>
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Advanced Imaging Facility
> Centre for Core Biotechnology Services University of Leicester
> www.le.ac.uk/advanced-imaging-facility
>
>
>
>
> -----Original Message-----
> From: Jacqueline Ross [mailto:[hidden email]]
> Sent: 15 May 2018 05:43
> To: [hidden email]
> Subject: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Hi everyone,
>
> I'm hoping that someone has done this before and has a solution in place that I can use or adapt.
>
> I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.
>
> However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.
>
> The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.
>
> I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU) School of Medical Sciences
> Faculty of Medical & Health Sciences The University of Auckland
> Private Bag 92019 Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI:  +64 9 923 7438
>
> Website:    http://www.auckland.ac.nz/biru
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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FW: Help wanted with time-lapse analysis - redirect to using stacks

Jacqueline Ross
In reply to this post by Jacqueline Ross
Hi all,

This is the binary image that I'm using to create the selections/ROIs.

Kind regards,

Jacqui

-----Original Message-----
From: Jacqueline Ross
Sent: Friday, 18 May 2018 6:36 p.m.
To: '[hidden email]' <[hidden email]>
Subject: RE: Help wanted with time-lapse analysis - redirect to using stacks

Hi Kees and Esteban (and anyone else trying to solve this!),

I've now tried using a macro written by Jeffrey Murphy. It relies on having two folders of images. So, I've put the binary mask images in Folder A and the raw images for the other channel in Folder B.

I still get different results when using the macro compared to manual  measurements. It must be something simple but I can't work it out. I have attached two images, hope they aren't too big to get through.

Here is my macro so far;

// Jeffrey N. Murphy
// @MurphysLab
// 2015-06-30

// This presumes that the images in Folder A and in Folder B are in the same order!

// Get a list of images in Folder A
image_directory_A = getDirectory("Choose Folder A"); file_list_A = getFileList(image_directory_A); image_list_A = newArray(0); image_count_A = 0; for(i=0; i<file_list_A.length; i++){
        if(endsWith(toLowerCase(file_list_A[i]),"tif")||endsWith(toLowerCase(file_list_A[i]),"png")){
                image_count_A++;
                image_list_A = Array.concat(image_list_A,file_list_A[i]);
        }
}

// Get a list of images in Folder B
image_directory_B = getDirectory("Choose Folder B"); file_list_B = getFileList(image_directory_B); image_list_B = newArray(0); image_count_B = 0; for(i=0; i<file_list_B.length; i++){
        if(endsWith(toLowerCase(file_list_B[i]),"tif")||endsWith(toLowerCase(file_list_B[i]),"png")){
                image_count_B++;
                image_list_B = Array.concat(image_list_B,file_list_B[i]);
        }
}

if(image_count_B == image_count_A){
        for(img_i=0; img_i<image_count_A; img_i++){
                open(image_directory_A + image_list_A[img_i]);
                image_A = getImageID();
                open(image_directory_B + image_list_B[img_i]);
                image_B = getImageID();
               
                // IMAGE A
                selectImage(image_A);
                // do what you need to make your ROI
                                //run("Threshold...");
setAutoThreshold("Moments dark");
run("Create Selection");
roiManager("Add");
                close();
               
                // IMAGE B
                selectImage(image_B);
                // measure what you need using the ROI roiManager("Show All");

                //run("Threshold...");
setAutoThreshold("Moments dark");
run("Set Measurements...", "area mean standard modal min integrated median skewness kurtosis area_fraction limit display redirect=None decimal=4"); roiManager("Select", 0); roiManager("Measure"); roiManager("Delete");
                close();
        }
}
               
Kind regards,

Jacqui
-----Original Message-----
From: Jacqueline Ross
Sent: Friday, 18 May 2018 6:31 p.m.
To: [hidden email]
Subject: RE: Help wanted with time-lapse analysis - redirect to using stacks

Hi Esteban,

Thanks for your reply. I tried your suggestion but can't seem to get it to work. The thing that troubles me is that I am able to get results using the Measure Stack macro but they don't match the results I get when I do the analysis manually.

Kind regards,

Jacqui

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez
Sent: Wednesday, 16 May 2018 10:44 a.m.
To: [hidden email]
Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqui,

Your approach works for me with the "Confocal Series" sample image but only if I check the "limit to threshold" box in Set Measurements and leave the red Threshold overlay activated on the binary stack.  The macro does scroll through the series.

I wonder if with your dataset you have to use "Stack.setFrame(n)" in your macro instead of "setSlice(n)", since it is a timelapse not a z-stack.

-Esteban

On Tue, May 15, 2018 at 3:18 PM, Jacqueline Ross <[hidden email]> wrote:

> Dear Kees,
>
> Thank you very much for that suggestion. I didn't know about that option. Only problem now is that I would like one result (summary) per slice rather than the individual values.
>
> I guess I could split the stack into individual images, combine the ROIs and then measure again.
>
> Kind regards,
>
> Jacqui
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Straatman, Kees (Dr.)
> Sent: Tuesday, 15 May 2018 7:16 p.m.
> To: [hidden email]
> Subject: Re: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Dear Jacqui,
>
> One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.
>
> Best wishes
>
> Kees
>
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Advanced Imaging Facility
> Centre for Core Biotechnology Services University of Leicester
> www.le.ac.uk/advanced-imaging-facility
>
>
>
>
> -----Original Message-----
> From: Jacqueline Ross [mailto:[hidden email]]
> Sent: 15 May 2018 05:43
> To: [hidden email]
> Subject: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Hi everyone,
>
> I'm hoping that someone has done this before and has a solution in place that I can use or adapt.
>
> I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.
>
> However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.
>
> The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.
>
> I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU) School of Medical Sciences
> Faculty of Medical & Health Sciences The University of Auckland
> Private Bag 92019 Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI:  +64 9 923 7438
>
> Website:    http://www.auckland.ac.nz/biru
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
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Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqueline Ross
In reply to this post by G. Esteban Fernandez
Hi everyone,

As suggested by Esteban, I have uploaded a sample data set on my Google drive. Currently, I'm sharing that with those who have already responded with some suggestions. If anyone else wants to take a look, please let me know.

The main issue I have is that after running the particle analyser (Analyze particles), I get multiple ROIs per time-point. I need to combine these ROIs into a single ROI so that I can get a measurement per slice (time-point).

If a solution is found, I will share it.

Kind regards,

Jacqui

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez
Sent: Wednesday, 16 May 2018 10:44 a.m.
To: [hidden email]
Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqui,

Your approach works for me with the "Confocal Series" sample image but only if I check the "limit to threshold" box in Set Measurements and leave the red Threshold overlay activated on the binary stack.  The macro does scroll through the series.

I wonder if with your dataset you have to use "Stack.setFrame(n)" in your macro instead of "setSlice(n)", since it is a timelapse not a z-stack.

-Esteban

On Tue, May 15, 2018 at 3:18 PM, Jacqueline Ross <[hidden email]> wrote:

> Dear Kees,
>
> Thank you very much for that suggestion. I didn't know about that option. Only problem now is that I would like one result (summary) per slice rather than the individual values.
>
> I guess I could split the stack into individual images, combine the ROIs and then measure again.
>
> Kind regards,
>
> Jacqui
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Straatman, Kees (Dr.)
> Sent: Tuesday, 15 May 2018 7:16 p.m.
> To: [hidden email]
> Subject: Re: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Dear Jacqui,
>
> One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.
>
> Best wishes
>
> Kees
>
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Advanced Imaging Facility
> Centre for Core Biotechnology Services University of Leicester
> www.le.ac.uk/advanced-imaging-facility
>
>
>
>
> -----Original Message-----
> From: Jacqueline Ross [mailto:[hidden email]]
> Sent: 15 May 2018 05:43
> To: [hidden email]
> Subject: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Hi everyone,
>
> I'm hoping that someone has done this before and has a solution in place that I can use or adapt.
>
> I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.
>
> However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.
>
> The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.
>
> I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU) School of Medical Sciences
> Faculty of Medical & Health Sciences The University of Auckland
> Private Bag 92019 Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI:  +64 9 923 7438
>
> Website:    http://www.auckland.ac.nz/biru
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
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Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqueline Ross
Hi everyone,

I may finally have got this working using Jeffrey Murphy's macro...

I have pasted the code below. If anyone has any other ideas or finds an issue with this, I would be grateful for the help.

// Get a list of images in Folder A
image_directory_A = getDirectory("Choose Folder A");
file_list_A = getFileList(image_directory_A);
image_list_A = newArray(0);
image_count_A = 0;
for(i=0; i<file_list_A.length; i++){
        if(endsWith(toLowerCase(file_list_A[i]),"tif")||endsWith(toLowerCase(file_list_A[i]),"png")){
                image_count_A++;
                image_list_A = Array.concat(image_list_A,file_list_A[i]);
        }
}

// Get a list of images in Folder B
image_directory_B = getDirectory("Choose Folder B");
file_list_B = getFileList(image_directory_B);
image_list_B = newArray(0);
image_count_B = 0;
for(i=0; i<file_list_B.length; i++){
        if(endsWith(toLowerCase(file_list_B[i]),"tif")||endsWith(toLowerCase(file_list_B[i]),"png")){
                image_count_B++;
                image_list_B = Array.concat(image_list_B,file_list_B[i]);
        }
}

if(image_count_B == image_count_A){
        for(img_i=0; img_i<image_count_A; img_i++){
                open(image_directory_A + image_list_A[img_i]);
                image_A = getImageID();
                open(image_directory_B + image_list_B[img_i]);
                image_B = getImageID();
               
                // IMAGE A
                selectImage(image_A);
                // do what you need to make your ROI
                                //run("Threshold...");
setAutoThreshold("Moments dark");
//run("Threshold...");
run("Create Selection");
roiManager("Add");
run("Set Measurements...", "area mean standard modal min integrated median skewness kurtosis area_fraction limit display redirect=None decimal=2");
roiManager("Measure");
                close();
               
                // IMAGE B
                selectImage(image_B);
                // measure what you need using the ROI
run("Set Measurements...", "area mean standard modal min integrated median skewness kurtosis area_fraction  display redirect=None decimal=2");
roiManager("Show All");
roiManager("Measure");
roiManager("Delete");
                close();
        }
}
               

Kind regards,

Jacqui

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Jacqueline Ross
Sent: Wednesday, 23 May 2018 11:55 a.m.
To: [hidden email]
Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks

Hi everyone,

As suggested by Esteban, I have uploaded a sample data set on my Google drive. Currently, I'm sharing that with those who have already responded with some suggestions. If anyone else wants to take a look, please let me know.

The main issue I have is that after running the particle analyser (Analyze particles), I get multiple ROIs per time-point. I need to combine these ROIs into a single ROI so that I can get a measurement per slice (time-point).

If a solution is found, I will share it.

Kind regards,

Jacqui

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez
Sent: Wednesday, 16 May 2018 10:44 a.m.
To: [hidden email]
Subject: Re: Help wanted with time-lapse analysis - redirect to using stacks

Jacqui,

Your approach works for me with the "Confocal Series" sample image but only if I check the "limit to threshold" box in Set Measurements and leave the red Threshold overlay activated on the binary stack.  The macro does scroll through the series.

I wonder if with your dataset you have to use "Stack.setFrame(n)" in your macro instead of "setSlice(n)", since it is a timelapse not a z-stack.

-Esteban

On Tue, May 15, 2018 at 3:18 PM, Jacqueline Ross <[hidden email]> wrote:

> Dear Kees,
>
> Thank you very much for that suggestion. I didn't know about that option. Only problem now is that I would like one result (summary) per slice rather than the individual values.
>
> I guess I could split the stack into individual images, combine the ROIs and then measure again.
>
> Kind regards,
>
> Jacqui
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Straatman, Kees (Dr.)
> Sent: Tuesday, 15 May 2018 7:16 p.m.
> To: [hidden email]
> Subject: Re: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Dear Jacqui,
>
> One option is to take the binary image and use Analyse particles to add these selection to the ROI Manager. Split the channels of the original image and take care that in the ROI Manager under More > Options... > 'Associate "Show All" ROIs with slices' is ticked. Select one of your split channel images and in the ROI Manager hit 'Measure' and the results table should pop-up with the measurements. Do the same for the second channel and that should be it.
>
> Best wishes
>
> Kees
>
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Advanced Imaging Facility
> Centre for Core Biotechnology Services University of Leicester
> www.le.ac.uk/advanced-imaging-facility
>
>
>
>
> -----Original Message-----
> From: Jacqueline Ross [mailto:[hidden email]]
> Sent: 15 May 2018 05:43
> To: [hidden email]
> Subject: Help wanted with time-lapse analysis - redirect to using
> stacks
>
> Hi everyone,
>
> I'm hoping that someone has done this before and has a solution in place that I can use or adapt.
>
> I have a time-lapse data set with two fluorescence markers. I've created a mask representing the overlap between the two markers using the Image Calculator so now have a binary stack to match the channel I want to measure. I want to measure gray values, area, integrated density, etc. defined by a threshold but only within the area where the two markers are collocated.
>
> However, when I try to do the measurements using the Redirect to: option and the Measure Stack macro, it seems like only the first image of the binary stack is used, not each corresponding slice.
>
> The problem I have is that there is movement of the labels between the time-points so I can't use a static ROI.
>
> I feel like this shouldn't be difficult but I can't get it working. I'd be very grateful for any assistance.
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit (BIRU) School of Medical Sciences
> Faculty of Medical & Health Sciences The University of Auckland
> Private Bag 92019 Auckland 1142, NEW ZEALAND
>
> Telephone: Ext 87438; DDI:  +64 9 923 7438
>
> Website:    http://www.auckland.ac.nz/biru
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
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--
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