Multi measure with moving ROI

[Kirk_Taylor]

Hi,
Firstly, apologies if there is already a thread for this, but I couldn't find one.

In my experiments I am measuring intracellular fluorescence of an ion-sensitive dye in response to stimulation by an agonist. These experiments are over a 10 minute time course with an image acquired every second (600 frames in total). I have used multi measure to assess the change in fluorescence and this works quite well on the whole (thank you ImageJ!!). However, my cells are non-adherent and during the course of the experiment some cells start to drift (slowly). This causes problems as some ROI's are no longer placed appropriately by the end of the of the experiment. Is there a way to get ImageJ to reposition the ROI for each frame?

Many thanks,
Kirk

[Menelaos Symeonides]

Hi Kirk,

I don't have a good answer for you, just throwing my hat in because I've
just been dealing with a very similar issue and would be interested to
hear what's out there. Because I had to get this done and couldn't wait
for a better solution to come up, I ended up doing the following:

1. Under Analyze - Set Measurements, I selected the measurements I was
interested in.

2. Under Analyze - Tools, I opened ROI Manager.

3. In each frame, I selected my ROI (in my case, a T cell I was tracking
in the brightfield channel to look for fluorescence appearing in the GFP
channel), making sure I had my channel of interest selected, and after
selecting each ROI I hit "t" to add the ROI to the ROI manager, and
advanced to the next timepoint.

4. After saving each ROI in each frame, I saved my set of ROIs within
ROI Manager, and then hit Measure under ROI Manager, which spit out a
nice table of my results.

Extremely time consuming, and after the 1500th ROI, rather mind-bending,
but I got my data.

Good luck,
Mel

On 10/22/2015 10:58 AM, Kirk_Taylor wrote:

> Hi,
> Firstly, apologies if there is already a thread for this, but I couldn't
> find one.
>
> In my experiments I am measuring intracellular fluorescence of an
> ion-sensitive dye in response to stimulation by an agonist. These
> experiments are over a 10 minute time course with an image acquired every
> second (600 frames in total). I have used multi measure to assess the change
> in fluorescence and this works quite well on the whole (thank you ImageJ!!).
> However, my cells are non-adherent and during the course of the experiment
> some cells start to drift (slowly). This causes problems as some ROI's are
> no longer placed appropriately by the end of the of the experiment. Is there
> a way to get ImageJ to reposition the ROI for each frame?
>
> Many thanks,
> Kirk
>
>
>
> --
> View this message in context: http://imagej.1557.x6.nabble.com/Multi-measure-with-moving-ROI-tp5014723.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]

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

[Krs5]

Hi Kirk,

Depending on the images you have you might be able to use something like the Image Stabilizer plugin (http://www.kangli.org/code/Image_Stabilizer.html) instead of changing the ROI.

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Advanced Imaging Facility
Centre for Core Biotechnology Services
University of Leicester
http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif



-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Kirk_Taylor
Sent: 22 October 2015 15:58
To: [hidden email]
Subject: Multi measure with moving ROI

Hi,
Firstly, apologies if there is already a thread for this, but I couldn't find one.

In my experiments I am measuring intracellular fluorescence of an ion-sensitive dye in response to stimulation by an agonist. These experiments are over a 10 minute time course with an image acquired every second (600 frames in total). I have used multi measure to assess the change in fluorescence and this works quite well on the whole (thank you ImageJ!!).
However, my cells are non-adherent and during the course of the experiment some cells start to drift (slowly). This causes problems as some ROI's are no longer placed appropriately by the end of the of the experiment. Is there a way to get ImageJ to reposition the ROI for each frame?

Many thanks,
Kirk



--
View this message in context: http://imagej.1557.x6.nabble.com/Multi-measure-with-moving-ROI-tp5014723.html
Sent from the ImageJ mailing list archive at Nabble.com.

--
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[Krs5]

Hi Kirk,

Realised that the Image Stabilizer plugin might not be the best option for slow drift correction. Some other options are:

StackReg + TurboReg:
Register Virtual Stack Slices
Linear Stack Alignment with SIFT
Elastic Alignment and Montage
BIJplugin (http://bij.isi.uu.nl/) allows ROI to align large images

Best wishes

Kees

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Straatman, Kees (Dr.)
Sent: 22 October 2015 16:28
To: [hidden email]
Subject: Re: Multi measure with moving ROI

Hi Kirk,

Depending on the images you have you might be able to use something like the Image Stabilizer plugin (http://www.kangli.org/code/Image_Stabilizer.html) instead of changing the ROI.

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Advanced Imaging Facility
Centre for Core Biotechnology Services
University of Leicester
http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif



-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Kirk_Taylor
Sent: 22 October 2015 15:58
To: [hidden email]
Subject: Multi measure with moving ROI

Hi,
Firstly, apologies if there is already a thread for this, but I couldn't find one.

In my experiments I am measuring intracellular fluorescence of an ion-sensitive dye in response to stimulation by an agonist. These experiments are over a 10 minute time course with an image acquired every second (600 frames in total). I have used multi measure to assess the change in fluorescence and this works quite well on the whole (thank you ImageJ!!).
However, my cells are non-adherent and during the course of the experiment some cells start to drift (slowly). This causes problems as some ROI's are no longer placed appropriately by the end of the of the experiment. Is there a way to get ImageJ to reposition the ROI for each frame?

Many thanks,
Kirk



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Sent from the ImageJ mailing list archive at Nabble.com.

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[Kirk_Taylor]

Thank you for the suggestions, I will give these a go and see if they solve the problem. I did try this a couple of years ago with some fewer frames and manually changed the ROI (similar to Mel's suggestion), however this does take a lot of time.

Best wishes,
Kirk

[Rebecca Keller]

Greetings,

I have dealt with this very same problem, and the workaround I used was to
consider the time-series as a third space dimension, then use the 3D object
finder (I think it was) to locate column-like ROI's, then evaluated these
ROI's at all time points. This requires the ROIs not to intersect, which
may or may not be possible, depending on your data. I can provide more info
if you need it.

Best Regards,

Jacob Keller

On Thu, Oct 22, 2015 at 12:11 PM, Kirk_Taylor <[hidden email]>
wrote:

> Thank you for the suggestions, I will give these a go and see if they solve
> the problem. I did try this a couple of years ago with some fewer frames
> and
> manually changed the ROI (similar to Mel's suggestion), however this does
> take a lot of time.
>
> Best wishes,
> Kirk
>
>
>
> --
> View this message in context:
> http://imagej.1557.x6.nabble.com/Multi-measure-with-moving-ROI-tp5014723p5014727.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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