Zeiss Definite Focus does nothing in "apply" mode

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Zeiss Definite Focus does nothing in "apply" mode

Jim Valcourt
Hi all,

***Summary***
I am unable to use Definite Focus with multiple stage positions because nothing happens when Definite Focus is invoked in "apply" mode. This happens in both the beta (2.0.0-beta3 20170409) and a recent stable release (1.4.22).

***More details***
I'm experiencing a problem similar to ones described previously on this list (here and here), but I was unable to find a resolution in those threads. Briefly, I'm following the directions at https://micro-manager.org/wiki/ZeissCAN29. For each XY position, I set the definite focus position in "Measure" mode by clicking the autofocus button in the main window, then marking that position in the position list. (Each position ends up with a unique ZeissDefiniteFocusOffset and an XYStage position.) I then set "ZeissDefiniteFocus-Focus Method" to "Apply" and start the acquisition. The LCD screen on the Definite Focus box displays "Off" throughout the acquisition and the focus drifts, so I conclude that the Definite Focus is not running properly.

I know that micro-manager is able to talk to the Definite Focus module because it works perfectly if I attempt to take a timelapse of a single position using "Last Position" mode instead of "Apply" mode.

I have a log file (in debug mode) from a session in which this behavior occurs, if that's helpful.

Thanks very much for your help!

Best,
Jim

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Re: Zeiss Definite Focus does nothing in "apply" mode

PhilippeP
Hi Jim,

We are doing HCS using MM2 lists and Zeiss DF.
What we do is:
Set a 3 point plane covering the areas of interest;
Create our list of positions , the extrapolated z being calculated by MM
from the 3 point plane defined above;
Set DF focus manually ("Measure") on a random position, then switch DF
to "Last Position";
Start the acquisition.
As long as the 3 point plane z positions are within the DF searching
distance, everything works smoothly.
I have no idea what "Apply" is for.

Hope that helps...
Philippe


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Re: Zeiss Definite Focus does nothing in "apply" mode

Jun Liao
Hi Philippe,

Thank you for your reply.

I just found that change the trigger mode from internal trigger to software trigger allows me to use lower exposure time.

Best,
Jun

On Tue, Apr 11, 2017 at 9:33 AM, Philippe Pognonec <[hidden email]> wrote:
Hi Jim,

We are doing HCS using MM2 lists and Zeiss DF.
What we do is:
Set a 3 point plane covering the areas of interest;
Create our list of positions , the extrapolated z being calculated by MM
from the 3 point plane defined above;
Set DF focus manually ("Measure") on a random position, then switch DF
to "Last Position";
Start the acquisition.
As long as the 3 point plane z positions are within the DF searching
distance, everything works smoothly.
I have no idea what "Apply" is for.

Hope that helps...
Philippe


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Re: Zeiss Definite Focus does nothing in "apply" mode

Jim Valcourt
In reply to this post by PhilippeP
Hi Philippe,

Thanks for your response. Unfortunately, my application requires imaging in slightly different planes in different wells; if I understand correctly what you're doing, I'm not sure that it will quite work without manually adding Z offsets to every position. I'll play around with it and see if I can get something to work.

Thanks,
Jim

On Tue, Apr 11, 2017 at 9:55 AM Philippe Pognonec <[hidden email]> wrote:
Hi Jim,

We are doing HCS using MM2 lists and Zeiss DF.
What we do is:
Set a 3 point plane covering the areas of interest;
Create our list of positions , the extrapolated z being calculated by MM
from the 3 point plane defined above;
Set DF focus manually ("Measure") on a random position, then switch DF
to "Last Position";
Start the acquisition.
As long as the 3 point plane z positions are within the DF searching
distance, everything works smoothly.
I have no idea what "Apply" is for.

Hope that helps...
Philippe


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Re: Zeiss Definite Focus does nothing in "apply" mode

M Fouet
Hi all,

We are experiencing the same issue as Jim: we cannot get the definite focus working if using Multi Acquisition and a list of positions.
Is there anyone who experienced the same issue and found a workaround?

Philippe > Thank you for sharing that technique with a 3 points plane. If you are implementing this via a script, would you mind sharing it?
It is still unclear to me what the sequence should be between the measure / apply or last measure method.

Thanks a lot for your help,

---
Marc Fouet
Postdoc fellow at the Rine Laboratory
Department of Molecular & Cell Biology
University of California, Berkeley
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Re: Zeiss Definite Focus does nothing in "apply" mode

Jim Valcourt
Hi Marc,

I actually ended up getting it to work; if I recall correctly, the key was to switch into "Apply" mode, update the DefiniteFocusOffset to the index you want, run full focus, switch into "Last Position" mode, and run full focus again. I'm at a conference for the next week, but I'll pass along the script I wrote once I get back.

Best,
Jim

On Fri, Jun 23, 2017 at 8:41 PM m.fouet <[hidden email]> wrote:
Hi all,

We are experiencing the same issue as Jim: we cannot get the definite focus
working if using Multi Acquisition and a list of positions.
Is there anyone who experienced the same issue and found a workaround?

Philippe > Thank you for sharing that technique with a 3 points plane. If
you are implementing this via a script, would you mind sharing it?
It is still unclear to me what the sequence should be between the measure /
apply or last measure method.

Thanks a lot for your help,

---
Marc Fouet
Postdoc fellow at the Rine Laboratory
Department of Molecular & Cell Biology
University of California, Berkeley



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Re: Zeiss Definite Focus does nothing in "apply" mode

M Fouet
Hi Jim,

Thanks for specifying the sequence. I tried to update the DefiniteFocusOffset with different indexes corresponding to the z positions I want, but after I switch to last position I still don't have the focus working.
And if you have time after, your script will be indeed highly appreciated!
Enjoy the conference,

Marc
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Re: Zeiss Definite Focus does nothing in "apply" mode

Karl Bellve-3

I got Definite Focus working with multiple positions (specifically a 96 well plate)  years back and I thought I posted the script on the list. So, search maybe >5 years ago.

On phone.... If I didn't post it, I can post it tomorrow.

Cheers Karl


On Mon, Jun 26, 2017, 7:06 PM M Fouet <[hidden email]> wrote:
Hi Jim,

Thanks for specifying the sequence. I tried to update the
DefiniteFocusOffset with different indexes corresponding to the z positions
I want, but after I switch to last position I still don't have the focus
working.
And if you have time after, your script will be indeed highly appreciated!
Enjoy the conference,

Marc



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View this message in context: http://micro-manager.3463995.n2.nabble.com/Zeiss-Definite-Focus-does-nothing-in-apply-mode-tp7588326p7588575.html
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Re: Zeiss Definite Focus does nothing in "apply" mode

M Fouet
Hi Karl,

The only code I found is in this message:

where apparently “Apply" is set systematically before “Last position” for every position.
As for the 96 well plate definite focus integration, I found this thread but no script:

I’ll keep searching but hopefully I should get by with this code and the HCS plugin.

Many thanks,

---
Marc

On 26 Jun 2017, at 16:29, Karl Bellve-3  wrote:

I got Definite Focus working with multiple positions (specifically a 96 well plate)  years back and I thought I posted the script on the list. So, search maybe >5 years ago.

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Re: Zeiss Definite Focus does nothing in "apply" mode

PhilippeP
In reply to this post by M Fouet
Sorry for the delay. For whatever reason, I could not post on this list anymore.

Philippe > Thank you for sharing that technique with a 3 points plane. If you are implementing this via a script, would you mind sharing it?
Hi Marc,
I use the HCS plugin for 3 points plane definition. No script, sorry.

It is still unclear to me what the sequence should be between the measure / apply or last measure method.
What we do is:
-Set focus manually
-In the "Focus" Window: set it to "Zeiss Definite Focus", and select "Measure"
-Click on the binocular icon to record that focus setting
-Switch Definite Focus to "Last Position"
-Start MDA (with of course Autofocus selected).

As long as the z of your positions in the recorded 3 point plane list are not too remote from the real focus, autofocus will work fine.
Hope that helps

Philippe
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Re: Zeiss Definite Focus does nothing in "apply" mode

Karl Bellve-3

I have attached a script from 2012 I must have used testing:

Hope it helps.

Cheers 

Karl Bellvé
Biomedical Imaging Group
Molecular Medicine
University of Massachusetts Medical School

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DefiniteFocus.bsh (6K) Download Attachment
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Re: Zeiss Definite Focus does nothing in "apply" mode

M Fouet
Hi,
Thanks a lot Karl, this was very helpful! I could set up the automation I wanted and understand better the “apply” step that is necessary to define the index. Works great!
---
Marc

> On 27 Jun 2017, at 07:31, Karl Bellve-3 [via Micro-Manager] <[hidden email]> wrote:
>
>
> I have attached a script from 2012 I must have used testing:
>
> Hope it helps.
>
> Cheers
>
> Karl Bellvé
> Biomedical Imaging Group
> Molecular Medicine
> University of Massachusetts Medical School
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