Multi-D Acquisition with Definite Focus

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Multi-D Acquisition with Definite Focus

nmankenbruck
Hello All,

I'm experiencing an issue using Definite Focus with micro-manager. We have an Axio Observer Z1 microscope with Definite Focus 1 installed. I have confirmed Definite Focus is functional from the touchpad attached to the stand.

I've been trying to perform multi-d acquisition with a single fluorescence channel at multiple positions of a slide with fixed cells for a longer period of time (~12 h). I selected my positions in X and Y and clicked the autofocus button in the main window. However, after the first acquisition, focus is quickly lost. Is there something I should be doing differently? I've already selected Definite Focus as the default autofocus drive in our configuration.

Thank you in advance for your assistance,

Nick
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Re: Multi-D Acquisition with Definite Focus

PhilippeP
Hi Nick

In our hands, Definite Focus is not working reliably on fixed slides. Do you have the same problem when working on fixed cells in PBS (in a plate)?

I assume you first did "Measure" in the autofocus menu to set up the DF on a manually focused position, and then selected "Last position" in the same menu to have DF run the autofocus on other positions? If yes, and if your slide is reasonably leveled, I unfortunately don't have other suggestions.

Philippe
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Re: Multi-D Acquisition with Definite Focus

Jim Valcourt-2
In reply to this post by nmankenbruck
Hi Nick,


MDA doesn’t seem to be able to handle multiple definite focus positions because the act of focusing requires both an “Apply” step to update the position and a “Last Position” step to actually set the focus. 

I had a similar issue and ended up writing a script to do it. Karl sent a script in that thread that should help clarify how to do it — if not, I can send you mine when I return from traveling. (I’m on mobile now, so I can’t check Karl’s script to confirm that it will work for you.)

Best,
Jim

On Tue, Jul 25, 2017 at 5:04 PM nmankenbruck <[hidden email]> wrote:
Hello All,

I'm experiencing an issue using Definite Focus with micro-manager. We have
an Axio Observer Z1 microscope with Definite Focus 1 installed. I have
confirmed Definite Focus is functional from the touchpad attached to the
stand.

I've been trying to perform multi-d acquisition with a single fluorescence
channel at multiple positions of a slide with fixed cells for a longer
period of time (~12 h). I selected my positions in X and Y and clicked the
autofocus button in the main window. However, after the first acquisition,
focus is quickly lost. Is there something I should be doing differently?
I've already selected Definite Focus as the default autofocus drive in our
configuration.

Thank you in advance for your assistance,

Nick



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Re: Multi-D Acquisition with Definite Focus

nmankenbruck
In reply to this post by PhilippeP
Hi Philippe,

Thanks for the response. I haven't tested fixed cells in a plate, only the slide. 

I did use "Measure" when I was finding each of the positions and initially was using "Apply" when I started the experiment. However, after looking at another forum post regarding a similar issue, I tried to use "Last position" and was still unsuccessful. I'm going to attempt to keep live cells in a plate in focus and see how that goes. 

Nick

On Thu, Jul 27, 2017 at 2:42 AM, PhilippeP <[hidden email]> wrote:
Hi Nick

In our hands, Definite Focus is not working reliably on fixed slides. Do you
have the same problem when working on fixed cells in PBS (in a plate)?

I assume you first did "Measure" in the autofocus menu to set up the DF on a
manually focused position, and then selected "Last position" in the same
menu to have DF run the autofocus on other positions? If yes, and if your
slide is reasonably leveled, I unfortunately don't have other suggestions.

Philippe



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