Re: [micro-manager-general] HCS plugin

[PEARSON M. <mat...@ig...>]

Hi Philippe and Kurt,

Thanks for the info, we will at least try a glass bottomed plate, not sure how flat they are but better than plastic.  Mark Tsuchida has also mentioned that some plate inserts may warp the plate through lateral forces, thought i'd mention for anyone else reading this.

Philippe, we are also using Zeiss and have definite focus although have had mixed success with it and didn't even try it for HCS.  Could you describe a little more how you use DF for HCS.  Where do you travel to to "measure" on the plate and how many offsets do you use?  When you travel to each first position in a grid and replace it in the xy list do you "measure" DF at the same time?  I noticed that when the xy positions are built into the list it does not take into account Z but i assume it does if you click replace as you say.

Thanks for the help,

Matt



 
--
Matt Pearson
Microscopy Facility
MRC Human Genetics Unit
IGMM
University of Edinburgh
Crewe Road
EH4 2XU



On 25 Jan 2016, at 18:24, Kurt Thorn <kur...@uc...>
 wrote:

> We have found that many plates are very far from flat - we have seen 
> plates where the well bottom differs by 200 um over the surface of the 
> plate.  This company makes plates that claim to be +/- 15 um over the 
> plate: 
> http://www.porvair-sciences.com/en/services-menu/life-sciences/glass-bottom-assay-plates/
> 
> We haven't tried them, but that might be a good starting point. Other 
> companies may have similar products; I don't know.
> 
> Kurt
> 
> On 1/25/2016 8:26 AM, 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
>> 
>> 
>> 
>> 
>> 
> 
> 
> -- 
> Kurt Thorn
> Associate Professor
> Director, Nikon Imaging Center
> http://thornlab.ucsf.edu/
> http://nic.ucsf.edu/blog/
> 
> 
> 
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