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Drift correction for live cell imaging

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Haitham Shaban

Drift correction for live cell imaging

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Hi all,

I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?

Thanks,
Haitham

Haitham A. Shaban
Chromatin and Gene Expression Group
Laboratoire de Biologie Moléculaire Eucaryote - CNRS
University of Toulouse

Jonathan Chubb

Re: Drift correction for live cell imaging

If you have a second spot, then you can use that as a reference, such as another locus- or the same locus in a diploid (see Wallace Marshall/John Sedat- Current Biology 1997, our paper in 2002). Some people use the SPB. Susan Gasser had a paper in 2001 (Heun et al, Science) where they didn't have a reference spot, but their spots were moving so much (yeast), it really didn't matter.

There is probably some fancy Fourier way to decompose the drift and diffusion components from the time series data, but I am not the person to ask.

From: Confocal Microscopy List <[hidden email]> on behalf of Haitham Shaban <[hidden email]>
Sent: 06 October 2016 20:33
To: [hidden email]
Subject: Drift correction for live cell imaging

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*****

Hi all,

I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?

Thanks,
Haitham

Haitham A. Shaban
Chromatin and Gene Expression Group
Laboratoire de Biologie Moléculaire Eucaryote - CNRS
University of Toulouse

Craig Brideau

Re: Drift correction for live cell imaging

XY drift is pretty straightforward. Do a rolling convolution on the images and note the offsets at which you get maximum correlation. That will be your offset between frames.

Craig

On Thu, Oct 6, 2016 at 1:52 PM, Chubb, Jonathan <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

If you have a second spot, then you can use that as a reference, such as another locus- or the same locus in a diploid (see Wallace Marshall/John Sedat- Current Biology 1997, our paper in 2002). Some people use the SPB. Susan Gasser had a paper in 2001 (Heun et al, Science) where they didn't have a reference spot, but their spots were moving so much (yeast), it really didn't matter.

There is probably some fancy Fourier way to decompose the drift and diffusion components from the time series data, but I am not the person to ask.

From: Confocal Microscopy List <[hidden email]> on behalf of Haitham Shaban <[hidden email]>
Sent: 06 October 2016 20:33
To: [hidden email]
Subject: Drift correction for live cell imaging

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU

lists.umn.edu

[hidden email]: listserv archives. confocalmicroscopy

Post images on http://www.imgur.com and include the link in your posting.

Imgur: The most awesome images on the Internet

www.imgur.com

Imgur is the best place to share and enjoy the most awesome images on the Internet. Every day, millions of people use Imgur to be entertained and inspired by funny ...

*****

Hi all,

I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?

Thanks,
Haitham

Haitham A. Shaban
Chromatin and Gene Expression Group
Laboratoire de Biologie Moléculaire Eucaryote - CNRS
University of Toulouse

Joshua Zachary Rappoport

Re: Drift correction for live cell imaging

We wrote software to do this automatically (see https://www.ncbi.nlm.nih.gov/pubmed/22044432)

Email me directly if you want us to share it with you

Josh

Joshua Z. Rappoport PhD

Director of the Center for Advanced Microscopy (CAM) and Nikon Imaging Center (NIC)

Northwestern University Feinberg School of Medicine
303 E. Chicago Avenue
Chicago, IL 60611

(312) 503-4140

http://cam.facilities.northwestern.edu/

http://nic.feinberg.northwestern.edu/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Thursday, October 06, 2016 4:29 PM
To: [hidden email]
Subject: Re: Drift correction for live cell imaging

XY drift is pretty straightforward. Do a rolling convolution on the images and note the offsets at which you get maximum correlation. That will be your offset between frames.

Craig

On Thu, Oct 6, 2016 at 1:52 PM, Chubb, Jonathan <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

If you have a second spot, then you can use that as a reference, such as another locus- or the same locus in a diploid (see Wallace Marshall/John Sedat- Current Biology 1997, our paper in 2002). Some people use the SPB. Susan Gasser had a paper in 2001 (Heun et al, Science) where they didn't have a reference spot, but their spots were moving so much (yeast), it really didn't matter.

There is probably some fancy Fourier way to decompose the drift and diffusion components from the time series data, but I am not the person to ask.

From: Confocal Microscopy List <[hidden email]> on behalf of Haitham Shaban <[hidden email]>
Sent: 06 October 2016 20:33
To: [hidden email]
Subject: Drift correction for live cell imaging

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU

lists.umn.edu

[hidden email]: listserv archives. confocalmicroscopy

Post images on http://www.imgur.com and include the link in your posting.

Imgur: The most awesome images on the Internet

www.imgur.com

Imgur is the best place to share and enjoy the most awesome images on the Internet. Every day, millions of people use Imgur to be entertained and inspired by funny ...

*****

Hi all,

I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?

Thanks,
Haitham

Haitham A. Shaban
Chromatin and Gene Expression Group
Laboratoire de Biologie Moléculaire Eucaryote - CNRS
University of Toulouse

G. Esteban Fernandez

Re: Drift correction for live cell imaging

In reply to this post by Haitham Shaban

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I use the following functions in FIJI ImageJ (free at http://fiji.sc):

For XY drift: Plugins > Registration > StackReg
For XYZ drift: Plugins > Registration > Correct 3D drift

-Esteban

On Thu, Oct 6, 2016 at 12:33 PM, Haitham Shaban
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?
>
> Thanks,
> Haitham
>
>
> Haitham A. Shaban
> Chromatin and Gene Expression Group
> Laboratoire de Biologie Moléculaire Eucaryote - CNRS
> University of Toulouse

Haitham Shaban

Re: Drift correction for live cell imaging

Thanks Esteban for your answer.

I think using StackReg Plugin for live cell imaging correction is incorrect, because it uses each slice as a template with respect to which the next slice, and then the correction goes in one direction. Since every thing inside the recorded video is moving so the correction by using StackReg will add wrong movement on the spot of interest for tracking.

Best,

Haitham

On Fri, Oct 7, 2016 at 12:20 AM, G. Esteban Fernandez <[hidden email]> wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I use the following functions in FIJI ImageJ (free at http://fiji.sc):

For XY drift: Plugins > Registration > StackReg
For XYZ drift: Plugins > Registration > Correct 3D drift

-Esteban

On Thu, Oct 6, 2016 at 12:33 PM, Haitham Shaban
<[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?
>
> Thanks,
> Haitham
>
>
> Haitham A. Shaban
> Chromatin and Gene Expression Group
> Laboratoire de Biologie Moléculaire Eucaryote - CNRS
> University of Toulouse

TSwayne

Re: Drift correction for live cell imaging

Hi Haitham,

As Jonathan mentioned, you need some other recognizable structure to use as a fiducial. Do you have other channels in your image?

If you have only one channel, maybe there is enough background labeling to segment the nucleus. You could then use that mask as a basis for automatic alignment — that way, the plugin will be looking at the whole nucleus and will not try to register the moving spot.

I believe you could then save the shift values and apply them to your original movie, so that you can track the spot relative to the nucleus.

If you could post an example image or short sequence it might inspire other solutions.

Hope this helps.

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

Theresa Swayne, Ph.D.

Manager

Confocal and Specialized Microscopy Shared Resource

Herbert Irving Comprehensive Cancer Center

Columbia University Medical Center

1130 St. Nicholas Ave., Room 222A

New York, NY 10032

Phone: 212-851-4613

[hidden email]

On Oct 7, 2016, at 12:10 AM, Haitham Shaban <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
Thanks Esteban for your answer.
I think using StackReg Plugin for live cell imaging correction is incorrect, because it uses each slice as a template with respect to which the next slice, and then the correction goes in one direction. Since every thing inside the recorded video is moving so the correction by using StackReg will add wrong movement on the spot of interest for tracking.

Best,

Haitham

On Fri, Oct 7, 2016 at 12:20 AM, G. Esteban Fernandez <[hidden email]> wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I use the following functions in FIJI ImageJ (free at http://fiji.sc):

For XY drift: Plugins > Registration > StackReg
For XYZ drift: Plugins > Registration > Correct 3D drift

-Esteban

On Thu, Oct 6, 2016 at 12:33 PM, Haitham Shaban
<[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?
>
> Thanks,
> Haitham
>
>
> Haitham A. Shaban
> Chromatin and Gene Expression Group
> Laboratoire de Biologie Moléculaire Eucaryote - CNRS
> University of Toulouse

Haitham Shaban

Re: Drift correction for live cell imaging

Hi Theresa,

Thanks for your helpful suggestion.I think the main problem is that I do not have anything is fixed in my image stack. I have background around the nucleus that could be used as a fiducial, but since it is a part of the cell so it also has movement. Also it suppose that both of them have different motion behaviour so if we went to relative the motions for the spot based on the mask came out from the background, it will add artifact on the motion of the spot. Please correct me if I am wrong.

I found it is forbidden to attach images and videos within this group; please find download a small video for this link spot of my interest inside the nucleus, that is could be good example for real problem :)

https://www.sendbigfiles.com/76e07de679d73aaca015d6d13602e3a9/

Best regards,

Haitham

On Fri, Oct 7, 2016 at 4:43 PM, Haitham Shaban <[hidden email]> wrote:

Hi Theresa,

Thanks for your helpful suggestion.I think the main problem is that I do not have anything is fixed in my image stack. I have background around the nucleus that could be used as a fiducial, but since it is a part of the cell so it also has movement. Also it suppose that both of them have different motion behaviour so if we went to relative the motions for the spot based on the mask came out from the background, it will add artifact on the motion of the spot. Please correct me if I am wrong.

Please find enclosed a small video for the spot of my interest inside the nucleus, that is could be good example for real problem :)

Best regards,
Haitham

On Fri, Oct 7, 2016 at 3:54 PM, Swayne, Theresa C. <[hidden email]> wrote:
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Haitham,

As Jonathan mentioned, you need some other recognizable structure to use as a fiducial. Do you have other channels in your image?

If you have only one channel, maybe there is enough background labeling to segment the nucleus. You could then use that mask as a basis for automatic alignment — that way, the plugin will be looking at the whole nucleus and will not try to register the moving spot.

I believe you could then save the shift values and apply them to your original movie, so that you can track the spot relative to the nucleus.

If you could post an example image or short sequence it might inspire other solutions.

Hope this helps.

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

Theresa Swayne, Ph.D.

Manager

Confocal and Specialized Microscopy Shared Resource

Herbert Irving Comprehensive Cancer Center

Columbia University Medical Center

1130 St. Nicholas Ave., Room 222A

New York, NY 10032

Phone: 212-851-4613

[hidden email]

On Oct 7, 2016, at 12:10 AM, Haitham Shaban <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
Thanks Esteban for your answer.
I think using StackReg Plugin for live cell imaging correction is incorrect, because it uses each slice as a template with respect to which the next slice, and then the correction goes in one direction. Since every thing inside the recorded video is moving so the correction by using StackReg will add wrong movement on the spot of interest for tracking.

Best,

Haitham

On Fri, Oct 7, 2016 at 12:20 AM, G. Esteban Fernandez <[hidden email]> wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I use the following functions in FIJI ImageJ (free at http://fiji.sc):

For XY drift: Plugins > Registration > StackReg
For XYZ drift: Plugins > Registration > Correct 3D drift

-Esteban

On Thu, Oct 6, 2016 at 12:33 PM, Haitham Shaban
<[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?
>
> Thanks,
> Haitham
>
>
> Haitham A. Shaban
> Chromatin and Gene Expression Group
> Laboratoire de Biologie Moléculaire Eucaryote - CNRS
> University of Toulouse

Julio Vazquez-2

Re: Drift correction for live cell imaging

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
Motion is only meaningful relative to a reference system. If you are tracking a single spot inside the nucleus, what is the reference system you are measuring the spot relative to? the slide may be moving relative to the camera; the cell may be moving relative to the slide; the nucleus may be moving relative to the cell... So I would say you first need to define precisely what motion you want to measure, and then choose and label a suitable reference object so that you can make your measurements. We faced a similar situation a few years ago when measuring chromosome motion, and our take was similar to that already mentioned of Wallace Marshall et al, namely to label two spots, and track their relative distance over time (Current Biology 11(16): 1227-1239. 2001). This gave us a measurement of the relative motion of two chromatin loci inside the nucleus, and was independent of the motion of everything else, including stage drift. If we assume that the two spots behave in a similar manner, then we can deduce the motion of each spot (or at least some aspects of it). We were lucky that the cells we were studying didn't have much motion themselves, so we could get a pretty good idea of the motion of each spot and verify that the two spots behaved in a very similar manner, but this is not always possible. Therefore, you need to take some time to think about the biological question you are interested in, which will lead you to a possible solution. For example, you could find the center of the nucleus, and use that as the reference (i.e. measure the distance between your spot and the center of the nucleus); or you could measure the shortest distance between your spot and the nuclear boundary; and so on.

Julio Vazquez

Fred Hutchinson Cancer Research Center

On Oct 7, 2016, at 7:52 AM, Haitham Shaban wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
Hi Theresa,

Thanks for your helpful suggestion.I think the main problem is that I do not have anything is fixed in my image stack. I have background around the nucleus that could be used as a fiducial, but since it is a part of the cell so it also has movement. Also it suppose that both of them have different motion behaviour so if we went to relative the motions for the spot based on the mask came out from the background, it will add artifact on the motion of the spot. Please correct me if I am wrong.

I found it is forbidden to attach images and videos within this group; please find download a small video for this link spot of my interest inside the nucleus, that is could be good example for real problem :)

https://www.sendbigfiles.com/76e07de679d73aaca015d6d13602e3a9/

Best regards,
Haitham

On Fri, Oct 7, 2016 at 4:43 PM, Haitham Shaban <[hidden email]> wrote:
Hi Theresa,

Thanks for your helpful suggestion.I think the main problem is that I do not have anything is fixed in my image stack. I have background around the nucleus that could be used as a fiducial, but since it is a part of the cell so it also has movement. Also it suppose that both of them have different motion behaviour so if we went to relative the motions for the spot based on the mask came out from the background, it will add artifact on the motion of the spot. Please correct me if I am wrong.

Please find enclosed a small video for the spot of my interest inside the nucleus, that is could be good example for real problem :)

Best regards,
Haitham

On Fri, Oct 7, 2016 at 3:54 PM, Swayne, Theresa C. <[hidden email]> wrote:
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Haitham,

As Jonathan mentioned, you need some other recognizable structure to use as a fiducial. Do you have other channels in your image?

If you have only one channel, maybe there is enough background labeling to segment the nucleus. You could then use that mask as a basis for automatic alignment — that way, the plugin will be looking at the whole nucleus and will not try to register the moving spot.

I believe you could then save the shift values and apply them to your original movie, so that you can track the spot relative to the nucleus.

If you could post an example image or short sequence it might inspire other solutions.

Hope this helps.

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

Theresa Swayne, Ph.D.

Manager

Confocal and Specialized Microscopy Shared Resource

Herbert Irving Comprehensive Cancer Center

Columbia University Medical Center

1130 St. Nicholas Ave., Room 222A

New York, NY 10032

Phone: 212-851-4613

[hidden email]

On Oct 7, 2016, at 12:10 AM, Haitham Shaban <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
Thanks Esteban for your answer.
I think using StackReg Plugin for live cell imaging correction is incorrect, because it uses each slice as a template with respect to which the next slice, and then the correction goes in one direction. Since every thing inside the recorded video is moving so the correction by using StackReg will add wrong movement on the spot of interest for tracking.

Best,

Haitham

On Fri, Oct 7, 2016 at 12:20 AM, G. Esteban Fernandez <[hidden email]> wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I use the following functions in FIJI ImageJ (free at http://fiji.sc):

For XY drift: Plugins > Registration > StackReg
For XYZ drift: Plugins > Registration > Correct 3D drift

-Esteban

On Thu, Oct 6, 2016 at 12:33 PM, Haitham Shaban
<[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> I tracking a single spot inside the nucleus for live cell imaging ( 50 sec duration) by spinning disk microscopy. Does anyone has suggestions for correcting the drift (lateral) in this case?
>
> Thanks,
> Haitham
>
>
> Haitham A. Shaban
> Chromatin and Gene Expression Group
> Laboratoire de Biologie Moléculaire Eucaryote - CNRS
> University of Toulouse

TSwayne

Re: Drift correction for live cell imaging

In reply to this post by Haitham Shaban

Hi Haitham,

I agree wholeheartedly with Julio Vazquez’s comments (included below). Your first task is to decide what frame of reference you will use to measure the motion of the spot. Nucleus, cell membrane, cell centroid, etc. It does not have to be a fixed point, it just needs to be a feature that you can identify in every frame.

The purpose of drift correction/registration is to align that feature in every timepoint so that it *becomes* fixed. Then you can measure the motion of other things in the movie relative to that feature. I hope that makes sense.

Looking at the sample image ex.tif, I see a bright moving nuclear spot, but I don’t see any drift of the whole nucleus or cell. So I’m not sure what you want to correct for in that movie.

Best,

Theresa

On Oct 7, 2016, at 10:52 AM, Haitham Shaban <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Theresa,

Thanks for your helpful suggestion.I think the main problem is that I do not have anything is fixed in my image stack. I have background around the nucleus that could be used as a fiducial, but since it is a part of the cell so it also has movement. Also it suppose that both of them have different motion behaviour so if we went to relative the motions for the spot based on the mask came out from the background, it will add artifact on the motion of the spot. Please correct me if I am wrong.

I found it is forbidden to attach images and videos within this group; please find download a small video for this link spot of my interest inside the nucleus, that is could be good example for real problem :)

https://www.sendbigfiles.com/76e07de679d73aaca015d6d13602e3a9/

Best regards,

Haitham

On Oct 7, 2016, at 11:58 AM, Julio Vazquez <[hidden email]> wrote:

Motion is only meaningful relative to a reference system. If you are tracking a single spot inside the nucleus, what is the reference system you are measuring the spot relative to? the slide may be moving relative to the camera; the cell may be moving relative to the slide; the nucleus may be moving relative to the cell... So I would say you first need to define precisely what motion you want to measure, and then choose and label a suitable reference object so that you can make your measurements. We faced a similar situation a few years ago when measuring chromosome motion, and our take was similar to that already mentioned of Wallace Marshall et al, namely to label two spots, and track their relative distance over time (Current Biology 11(16): 1227-1239. 2001). This gave us a measurement of the relative motion of two chromatin loci inside the nucleus, and was independent of the motion of everything else, including stage drift. If we assume that the two spots behave in a similar manner, then we can deduce the motion of each spot (or at least some aspects of it). We were lucky that the cells we were studying didn't have much motion themselves, so we could get a pretty good idea of the motion of each spot and verify that the two spots behaved in a very similar manner, but this is not always possible. Therefore, you need to take some time to think about the biological question you are interested in, which will lead you to a possible solution. For example, you could find the center of the nucleus, and use that as the reference (i.e. measure the distance between your spot and the center of the nucleus); or you could measure the shortest distance between your spot and the nuclear boundary; and so on.

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

Theresa Swayne, Ph.D.

Manager

Confocal and Specialized Microscopy Shared Resource

Herbert Irving Comprehensive Cancer Center

Columbia University Medical Center

1130 St. Nicholas Ave., Room 222A

New York, NY 10032

Phone: 212-851-4613

[hidden email]

mcammer

Re: Drift correction for live cell imaging

An extreme point about alignment over time.

https://www.flickr.com/photos/mcammer/3623501576/ This movie is an intermediate step in aligning a sperm that was swimming in a circle; the subsequent step (don’t know where the movie is) aligned the sperm head so wasn’t moving at all, only tail was, and this was used to measure the tail movement.

The image on the top https://www.flickr.com/photos/mcammer/3623501756/ is a projection of the original movie and the image on the bottom is a projection of the aligned image.

=========================================================================

Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center

Cell: 914-309-3270 Office: Skirball 2^nd Floor main office, back right

http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Swayne, Theresa C.
Sent: Monday, October 10, 2016 2:45 PM
To: [hidden email]
Subject: Re: Drift correction for live cell imaging

Hi Haitham,

Looking at the sample image ex.tif, I see a bright moving nuclear spot, but I don’t see any drift of the whole nucleus or cell. So I’m not sure what you want to correct for in that movie.

Best,

Theresa

On Oct 7, 2016, at 10:52 AM, Haitham Shaban <[hidden email]> wrote:

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Hi Theresa,

Thanks for your helpful suggestion.I think the main problem is that I do not have anything is fixed in my image stack. I have background around the nucleus that could be used as a fiducial, but since it is a part of the cell so it also has movement. Also it suppose that both of them have different motion behaviour so if we went to relative the motions for the spot based on the mask came out from the background, it will add artifact on the motion of the spot. Please correct me if I am wrong.

I found it is forbidden to attach images and videos within this group; please find download a small video for this link spot of my interest inside the nucleus, that is could be good example for real problem :)

https://www.sendbigfiles.com/76e07de679d73aaca015d6d13602e3a9/

Best regards,

Haitham

On Oct 7, 2016, at 11:58 AM, Julio Vazquez <[hidden email]> wrote:

Motion is only meaningful relative to a reference system. If you are tracking a single spot inside the nucleus, what is the reference system you are measuring the spot relative to? the slide may be moving relative to the camera; the cell may be moving relative to the slide; the nucleus may be moving relative to the cell... So I would say you first need to define precisely what motion you want to measure, and then choose and label a suitable reference object so that you can make your measurements. We faced a similar situation a few years ago when measuring chromosome motion, and our take was similar to that already mentioned of Wallace Marshall et al, namely to label two spots, and track their relative distance over time (Current Biology 11(16): 1227-1239. 2001). This gave us a measurement of the relative motion of two chromatin loci inside the nucleus, and was independent of the motion of everything else, including stage drift. If we assume that the two spots behave in a similar manner, then we can deduce the motion of each spot (or at least some aspects of it). We were lucky that the cells we were studying didn't have much motion themselves, so we could get a pretty good idea of the motion of each spot and verify that the two spots behaved in a very similar manner, but this is not always possible. Therefore, you need to take some time to think about the biological question you are interested in, which will lead you to a possible solution. For example, you could find the center of the nucleus, and use that as the reference (i.e. measure the distance between your spot and the center of the nucleus); or you could measure the shortest distance between your spot and the nuclear boundary; and so on.

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

Theresa Swayne, Ph.D.

Manager

Confocal and Specialized Microscopy Shared Resource

Herbert Irving Comprehensive Cancer Center

Columbia University Medical Center

1130 St. Nicholas Ave., Room 222A

New York, NY 10032

Phone: 212-851-4613

[hidden email]

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