Grid like artifact

Bayesian analysis of blinking and bleaching

Grid like artifact

Postby Sam » Tue Mar 19, 2013 12:57 pm

Hi there,

I am imaging a fairly small structure - I suspect it is a ring about 100nm to 200nm in diameter. I am trying to use threeB to get better data on its shape. When I run threeB on my data, I get the grid like artifact that you mention in one of your papers.

Do you have any advice on how to resolve this?

Thanks,

Sam
Sam
 
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Re: Grid like artifact

Postby susancox » Wed Mar 20, 2013 5:33 pm

Hi Sam,

Usually the grid artifact appears when there is insufficient data to do a good reconstruction. However, it can sometimes happen for other reasons. To help diagnose, there are a few things it would be helpful to know:
1) What fluorophore are you using? Is your experiment in fixed cells or live cells?
2) What is your light source, camera and exposure time?
3) When you do the reconstruction, are you using the ImageJ plugin to plot your data or are you using another plotting program?
susancox
 
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Re: Grid like artifact

Postby Sam » Thu Mar 28, 2013 2:09 pm

1) I am using YFP tagged protein in unfixed, detergent treated cytoskeletons
2) standard mercury vapour arc lamp, Andor iXon Ultra 897 EMCCD camera, exposure time ranging from 30ms to 100ms
3) I am using the ImageJ plugin.

The initial number of spots seems to make a big difference to how the final output looks. When I reduce this value, the grid disappears, although I think if it reduced too much then this is also adversely affecting the final output. Is there a way of estimating correct initial number of spots?
Sam
 
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Joined: Fri Mar 01, 2013 12:33 am

Re: Grid like artifact

Postby susancox » Tue Apr 02, 2013 2:25 pm

Hi Sam,

The initial number of spots should not affect the final output significantly, provided that 3B runs for enough iterations. Putting down too many spots can cause two effects: firstly it makes the iterations take longer and secondly you may need more iterations for the output to stabilise. As a result, it's generally better to err on the side of too few spots rather than too many.

The plugin attempts to estimate the initial number of spots. Maybe this is what is causing problems - the estimate is based on area so if your density is low it may not be optimal. Could you tell me approximately how large is the area you are analysing and how many iterations has it run for when you terminate it?
susancox
 
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Re: Grid like artifact

Postby Sam » Mon Apr 08, 2013 9:54 pm

Hi there Susan,

Apologies for the delayed reply - I was re-running the program with more iterations to ensure it had come to convergence (it takes some time!).

I have re-run the program using a variable number of starting spots (1 - 64) for between 500 and 900 iterations, to make sure it had come to convergence. The artifact remains on the outputs with more than 20 spots.

The starting area is 637 pixels in this case, with 160nm per pixel and 50ms exposure.

In case it is of any help, I have pasted links to the relevant data below.

This is a public dropbox link to the input file: http://db.tt/bt3hSY1r
This is a dropbox link to the ROI used: http://db.tt/pELqlOaa

The below are dropbox public links to the reconstructions:
1 spot: http://db.tt/Xr8NeRLA
5 spots: http://db.tt/C9jNP5Kp
10 spots: http://db.tt/voVXIQdF
15 spots: http://db.tt/ObZGAKTK
20 spots: http://db.tt/0hep1jCZ
30 spots: http://db.tt/z1gK8UUN
40 spots: http://db.tt/HJiyy67R
64 spots: http://db.tt/y9KbzAYs

Many thanks for your help!

Sam
Sam
 
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Joined: Fri Mar 01, 2013 12:33 am

Re: Grid like artifact

Postby morales » Wed Jul 24, 2013 3:45 pm

Sam wrote:Hi there Susan,
The artifact remains on the outputs with more than 20 spots.
Sam


Hi Susan and Sam,

Could any of you post a picture of this grid artifact? The dropbox links are not public, are they?

Thanks in advance
morales
 
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Joined: Tue Jul 23, 2013 1:24 pm


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