Re: [micro-manager-general] HCS plugin

[<Phi...@un...>]

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