Re: [micro-manager-general] HCS plugin

[Philippe P. <pog...@un...>]

Hi Matt,
We are happily using the HCS plugin with Micro-Manager.
We at first had a similar problem when changing well. The best solution 
we found is the following combination:

- Stable temperature during the whole process (with microscope 
temperature already stabilized at start);
- Bottom "glass" plate;
- Definite Focus activated. Don't forget to set it on "Last position" 
after "Measure" has been set. We use Zeiss. Any similar system should do;
- Manually verify the first position of the chosen grid in EACH well 
(yes, it's a pain...), and do "Replace" for each first position. This 
way, the z is memorized, and acts as a good starting point for the 
Definite Focus.
- Don't forget to save the position list, in case of MM crash...

By the way, it's too bad HCS plugin cannot set grids in a circular 
pattern, to avoid "wasting" cells that cannot be included in a square 
grid (our wells are round).
Philippe


On 01/25/2016 05:26 PM, PEARSON Matthew wrote:
> Hi all,
>
> I was wondering how many of you use the High Content Screening plugin within Micro-Manager?  And whether you can auto-focus reliably? I have a plastic 96 well plate and struggling to achieve consistent focus using either the 3 point auto focus or JAF (H & P) methods.  There seems to be considerable Z offset between the wells and the multi-well plate insert and stage has no grub screws for adjusting any tilt.  I am imaging at 10x so perhaps at the limit of what the 3 point interpolation focus can deal with.  I have tested JAF (H&P) be defocussing the live image and trying different z steps and i have been able to get it to work sometimes like this but as soon as i set it to capture all the imaging sites, many are out of focus.  We may try using a glass bottomed plate which should at least decrease the error inherent in the plate itself.  I just wondered how much this is being used by the community for HCS.
>
> Thanks,
>
> Matt
>
>
> --
> Matt Pearson
> Microscopy Facility
> MRC Human Genetics Unit
> IGMM
> University of Edinburgh
> Crewe Road
> EH4 2XU
>
>
>
>
>

-- 
Philippe Pognonec, Ph.D.
Directeur de Recherche CNRS
Transporteurs en Imagerie et Radiotherapie en Oncologie, CEA
Faculté de Médecine, Université de Nice
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06107 Nice Cedex 2
France

Tel/fax 33 493 37 77 14