First frame in MDA stack is wrong

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First frame in MDA stack is wrong

Peter Braubach

Hi all,

 

I recently experienced a strange behaviour of MM during MDA with z-stacks and multiple channels (3).

The frame taken by the camera is one-“off”. This results in the first image of the stack being from the previous channel (for example DAPI nuclear stain in the first image of the FITC channel, and so on).

I cannot find any camera configuration that solves this issue completely.

 

MM Version is 1.4.23 20180121

The Camera is an Point Grey GRAS-14-S5M

Camera device properties: https://www.dropbox.com/s/fktxevbebzs59ot/cameraconfig.jpg?dl=0

 

Sometimes this does not happen, I have larger multi position scans with this error occurring roughly in 50% of the images.

Recently it stuck and I cannot reproduce this intermittent behaviour any more.

 

Any help or suggestions what to try next would be greatly appreciated.

 

Peter


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Re: First frame in MDA stack is wrong

centswu
Hi Peter,

What’s your exposure time during this situation?



Hi all,

 

I recently experienced a strange behaviour of MM during MDA with z-stacks and multiple channels (3).

The frame taken by the camera is one-“off”. This results in the first image of the stack being from the previous channel (for example DAPI nuclear stain in the first image of the FITC channel, and so on).

I cannot find any camera configuration that solves this issue completely.

 

MM Version is 1.4.23 20180121

The Camera is an Point Grey GRAS-14-S5M

Camera device properties: https://www.dropbox.com/s/fktxevbebzs59ot/cameraconfig.jpg?dl=0

 

Sometimes this does not happen, I have larger multi position scans with this error occurring roughly in 50% of the images.

Recently it stuck and I cannot reproduce this intermittent behaviour any more.

 

Any help or suggestions what to try next would be greatly appreciated.

 

Peter

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Re: First frame in MDA stack is wrong

Peter Braubach
Exposure times are 4ms, 120ms and 80ms.
It also happens with longer (600ms, 800ms) exposure times.

Peter



Sent from Samsung tablet


-------- Original message --------
From: cents wu <[hidden email]>
Date: 04/05/2018 03:14 (GMT+01:00)
To: Micro-Manager General <[hidden email]>
Subject: Re: [micro-manager-general] First frame in MDA stack is wrong

Hi Peter,

What’s your exposure time during this situation?



Hi all,

 

I recently experienced a strange behaviour of MM during MDA with z-stacks and multiple channels (3).

The frame taken by the camera is one-“off”. This results in the first image of the stack being from the previous channel (for example DAPI nuclear stain in the first image of the FITC channel, and so on).

I cannot find any camera configuration that solves this issue completely.

 

MM Version is 1.4.23 20180121

The Camera is an Point Grey GRAS-14-S5M

Camera device properties: https://www.dropbox.com/s/fktxevbebzs59ot/cameraconfig.jpg?dl=0

 

Sometimes this does not happen, I have larger multi position scans with this error occurring roughly in 50% of the images.

Recently it stuck and I cannot reproduce this intermittent behaviour any more.

 

Any help or suggestions what to try next would be greatly appreciated.

 

Peter

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Re: First frame in MDA stack is wrong

Sam Lord
In reply to this post by Peter Braubach
Peter Braubach wrote
> This results in the first image of the stack being from the previous
> channel (for example DAPI nuclear stain in the first image of the FITC
> channel, and so on).

I have observed similar issues lately with our Andor iXon cameras. It
definitely happens if there is any sequencing involved (e.g. channels being
pre-loaded into an Arduino). The first two frames are duplicates. For
example, if I sequence 3 channels with zero delay ("burst"), the three
channels looks different (although I don't know about the very first frame).
If I add a 5 second delay, the first 2 channels are duplicates and the 3rd
channel is either correct or maybe out of order (actually the second
channel). I can correct this by changing the exposure times of the 3
channels to force the MDA to not use sequencing.

Sorry, I have more experimenting to come up with a complete description of
the error. I just wanted to say that I too am seeing an issue.

Sam



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Re: First frame in MDA stack is wrong

Peter Braubach
Hello all,

 this weekend I have tried to replicate the problem for some time, but I could neither come up with a consistent behavior nor adequate instructions for reproduction.

 I switched the setup to a different camera (original was a point grey Grasshopper 1, I switched to a grasshopper 2 I had lying around and this camera did not show the issue. After switching back to the original GS1 the issue was not replicable for some time before suddenly coming up again. This is really unnerving.
If someone has suggestions what to do next to track down the issue I am happy to try.

Kind regards
Peter

--
Dr. med. Peter Braubach
Koordinator Lungengewebebank
Arbeitsgruppe Lungenforschung

Institut für Pathologie
Medizinische Hochschule Hannover
OE 5110, Carl-Neuberg-Str. 1, 30625 Hannover, Deutschland
Tel.: +49 511 532-8494
[hidden email]
www.mh-hannover.de


-----Ursprüngliche Nachricht-----
Von: Sam Lord [mailto:[hidden email]]
Gesendet: Samstag, 5. Mai 2018 01:44
An: [hidden email]
Betreff: Re: [micro-manager-general] First frame in MDA stack is wrong

Peter Braubach wrote
> This results in the first image of the stack being from the previous
> channel (for example DAPI nuclear stain in the first image of the FITC
> channel, and so on).

I have observed similar issues lately with our Andor iXon cameras. It
definitely happens if there is any sequencing involved (e.g. channels being
pre-loaded into an Arduino). The first two frames are duplicates. For
example, if I sequence 3 channels with zero delay ("burst"), the three
channels looks different (although I don't know about the very first frame).
If I add a 5 second delay, the first 2 channels are duplicates and the 3rd
channel is either correct or maybe out of order (actually the second
channel). I can correct this by changing the exposure times of the 3
channels to force the MDA to not use sequencing.

Sorry, I have more experimenting to come up with a complete description of
the error. I just wanted to say that I too am seeing an issue.

Sam



--
Sent from: http://micro-manager.3463995.n2.nabble.com/

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Re: First frame in MDA stack is wrong

Peter Braubach
Hello all,
 
I was able to fix the problem by setting the trigger mode from "Software" to "Internal" on the Grashopper 1.


Kind regards
Peter
-----Ursprüngliche Nachricht-----
Von: [hidden email] [mailto:[hidden email]]
Gesendet: Montag, 7. Mai 2018 12:32
An: [hidden email]
Betreff: Re: [micro-manager-general] First frame in MDA stack is wrong

Hello all,

 this weekend I have tried to replicate the problem for some time, but I could neither come up with a consistent behavior nor adequate instructions for reproduction.

 I switched the setup to a different camera (original was a point grey Grasshopper 1, I switched to a grasshopper 2 I had lying around and this camera did not show the issue. After switching back to the original GS1 the issue was not replicable for some time before suddenly coming up again. This is really unnerving.
If someone has suggestions what to do next to track down the issue I am happy to try.

Kind regards
Peter

--
Dr. med. Peter Braubach
Koordinator Lungengewebebank
Arbeitsgruppe Lungenforschung

Institut für Pathologie
Medizinische Hochschule Hannover
OE 5110, Carl-Neuberg-Str. 1, 30625 Hannover, Deutschland
Tel.: +49 511 532-8494
[hidden email]
www.mh-hannover.de


-----Ursprüngliche Nachricht-----
Von: Sam Lord [mailto:[hidden email]]
Gesendet: Samstag, 5. Mai 2018 01:44
An: [hidden email]
Betreff: Re: [micro-manager-general] First frame in MDA stack is wrong

Peter Braubach wrote
> This results in the first image of the stack being from the previous
> channel (for example DAPI nuclear stain in the first image of the FITC
> channel, and so on).

I have observed similar issues lately with our Andor iXon cameras. It
definitely happens if there is any sequencing involved (e.g. channels being
pre-loaded into an Arduino). The first two frames are duplicates. For
example, if I sequence 3 channels with zero delay ("burst"), the three
channels looks different (although I don't know about the very first frame).
If I add a 5 second delay, the first 2 channels are duplicates and the 3rd
channel is either correct or maybe out of order (actually the second
channel). I can correct this by changing the exposure times of the 3
channels to force the MDA to not use sequencing.

Sorry, I have more experimenting to come up with a complete description of
the error. I just wanted to say that I too am seeing an issue.

Sam



--
Sent from: http://micro-manager.3463995.n2.nabble.com/

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