Re: [micro-manager-general] HCS plugin

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

Thanks Philippe thats very helpful will give it a try.

thanks,

Matt


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



On 26 Jan 2016, at 10:17, <Phi...@un...>
 wrote:

> 
> 
> On 26/01/2016 10:12, PEARSON Matthew wrote:
>> Where do you travel to to "measure" on the plate and how many offsets do you use?
> We use Ibidi glass bottom 24 plates, that have a pretty stable bottom 
> thickness, while not being perfectly flat, which turns out not to be a 
> big problem. We usually work with a 40x objective. For DF setting, with 
> simply select Zeiss DF through the Autofocus wrench icon button,  select 
> "Measure", manually focus on the cells anywhere on the plate and record 
> that setting by clicking on the binoculars. We then set the DF to "Last 
> position", and that's it (we close the autofocus properties windows at 
> that point).
> The problem we had was that DF cannot "find" the focus on another spot 
> of the plate if it is too much off (in term of z). This is why we,
>> 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.
> using the Stage Position List "Go to" button, go to each first position 
> of each grid created with the HCS plugin (always called 
> "Well_Address_Site_0". We do a rough focusing by hand, we click on the 
> focusing button (binoculars) to get a perfect focus, and do "Replace" in 
> the Stage Position List. That way, the z is recorded and will be used 
> when acquiring. This will give a reliable first z at the beginning each 
> grid.
> To avoid getting out of the DF range, we tend to DF at every frame. But 
> we successfully acquired images by skipping some frames in the Autofocus 
> option of the MDA when in a rush with many images to acquire. But be 
> aware that sometimes DF gets off, and images stay blurry till next grid...
> 
> Hope that helps. Good luck!
> Philippe
> 
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