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Re: HCS plugin

Philippe.POGNONEC


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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Re: HCS plugin

PEARSON Matthew
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, <[hidden email]>
 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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Re: HCS plugin

Nico Stuurman-2
In reply to this post by PEARSON Matthew
HI Matt,

On 1/26/16 1:12 AM, PEARSON Matthew wrote:
Hi Philippe and Kurt,

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.

Just for clarification: the user determines herself which axes are recorded in the Position List using the "Use" checkboxes in the bottom of the list. If you had not checked the Z-drive previously and marked a number of positions, then check the Z-drive and go to those positions and replace them, the net effect will be that you now include the Z-drive in the position. 

Best,

Nico



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Re: HCS plugin

PEARSON Matthew
Hi Nico,

The zeiss z focus axis check box was selected but when the xy position list was built from the HCS plugin, the list didn't seem to have the z position as well by default.

Thanks,

Matt

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



On 26 Jan 2016, at 17:39, Nico Stuurman <[hidden email]> wrote:

HI Matt,

On 1/26/16 1:12 AM, PEARSON Matthew wrote:
Hi Philippe and Kurt,

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.

Just for clarification: the user determines herself which axes are recorded in the Position List using the "Use" checkboxes in the bottom of the list. If you had not checked the Z-drive previously and marked a number of positions, then check the Z-drive and go to those positions and replace them, the net effect will be that you now include the Z-drive in the position. 

Best,

Nico


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Re: HCS plugin

Nico Stuurman-2

On 1/27/16 7:09 AM, PEARSON Matthew wrote:
The zeiss z focus axis check box was selected but when the xy position list was built from the HCS plugin, the list didn't seem to have the z position as well by default.

Ah yes, the HCS plugin does not look at those settings.  However, it will include the z-drive coordinates if you first perform the 3-point calibration (bottom right of the HCS plugin).  Did you try that?

Best,

Nico



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Re: HCS plugin

Damir Sudar
Hi Nico,

Looks like recently there are quite a few questions about the HCS Site Generator. We actually use that extensively as well and I also have a question: I'm upgrading to 1.4.22 and noticed that you had changed the acquisition pathing from the original typewriter style to the snakelike style. That is probably great for most folks but alas for us, that is actually quite detrimental. Is there an easy way for me to change that back to typewriter style?

For background: we print a high density microarray pattern of microenvironment spots (collagen with different microenvironment proteins) and we use the HCS plugin to map that array of locations in order to image the spots in the same sequence as they are printed. So the mapping is easy. With the snakestyle, that mapping becomes suddenly difficult and very likely to get messed up.

Thanks,
- Damir

On 1/27/2016 10:37 AM, Nico Stuurman wrote:

<unrelated questions and answers about the HCS plugin.>

Best,

Nico




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One Cyclotron Road, MS 977, Berkeley, CA 94720, USA
T: 510/486-5346 - F: 510/486-5586 - E: [hidden email]
WWW: http://www.lbl.gov/lifesciences/labs/sudar_lab.html

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Re: HCS plugin

Mark Tsuchida-3
Hi Damir,

On Wed, Feb 3, 2016 at 11:15 AM, Damir Sudar <[hidden email]> wrote:
Looks like recently there are quite a few questions about the HCS Site Generator. We actually use that extensively as well and I also have a question: I'm upgrading to 1.4.22 and noticed that you had changed the acquisition pathing from the original typewriter style to the snakelike style. That is probably great for most folks but alas for us, that is actually quite detrimental. Is there an easy way for me to change that back to typewriter style?

For background: we print a high density microarray pattern of microenvironment spots (collagen with different microenvironment proteins) and we use the HCS plugin to map that array of locations in order to image the spots in the same sequence as they are printed. So the mapping is easy. With the snakestyle, that mapping becomes suddenly difficult and very likely to get messed up.

Thanks for the suggestion. This sounds reasonable and I created a ticket for it.

Best,
Mark 

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Re: HCS plugin

Nico Stuurman-2

On 2/3/16 1:45 PM, Mark Tsuchida wrote:
Hi Damir,

On Wed, Feb 3, 2016 at 11:15 AM, Damir Sudar <[hidden email]> wrote:
Looks like recently there are quite a few questions about the HCS Site Generator. We actually use that extensively as well and I also have a question: I'm upgrading to 1.4.22 and noticed that you had changed the acquisition pathing from the original typewriter style to the snakelike style. That is probably great for most folks but alas for us, that is actually quite detrimental. Is there an easy way for me to change that back to typewriter style?

For background: we print a high density microarray pattern of microenvironment spots (collagen with different microenvironment proteins) and we use the HCS plugin to map that array of locations in order to image the spots in the same sequence as they are printed. So the mapping is easy. With the snakestyle, that mapping becomes suddenly difficult and very likely to get messed up.

Thanks for the suggestion. This sounds reasonable and I created a ticket for it.

Since I created the snake pattern I felt responsible and added an option to not use the snake pattern.  Regretfully, the UI code of the plugin is kind of a mess and I had to rework quite a bit to bring some sanity to it.  Since I do not have much time, problems may have slipped in, so please test at your earliest convenience and let me know of problems.  The change should be in the latest nightly build (may need to be back-ported into 2.0).

Best,

Nico



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Re: HCS plugin

Chris Weisiger


On Wed, Feb 3, 2016 at 9:26 PM, Nico Stuurman <[hidden email]> wrote:

On 2/3/16 1:45 PM, Mark Tsuchida wrote:
Hi Damir,

On Wed, Feb 3, 2016 at 11:15 AM, Damir Sudar <[hidden email]> wrote:
Looks like recently there are quite a few questions about the HCS Site Generator. We actually use that extensively as well and I also have a question: I'm upgrading to 1.4.22 and noticed that you had changed the acquisition pathing from the original typewriter style to the snakelike style. That is probably great for most folks but alas for us, that is actually quite detrimental. Is there an easy way for me to change that back to typewriter style?

For background: we print a high density microarray pattern of microenvironment spots (collagen with different microenvironment proteins) and we use the HCS plugin to map that array of locations in order to image the spots in the same sequence as they are printed. So the mapping is easy. With the snakestyle, that mapping becomes suddenly difficult and very likely to get messed up.

Thanks for the suggestion. This sounds reasonable and I created a ticket for it.

Since I created the snake pattern I felt responsible and added an option to not use the snake pattern.  Regretfully, the UI code of the plugin is kind of a mess and I had to rework quite a bit to bring some sanity to it.  Since I do not have much time, problems may have slipped in, so please test at your earliest convenience and let me know of problems.  The change should be in the latest nightly build (may need to be back-ported into 2.0).

This has now been done for 2.0 as well.

-Chris

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Re: HCS plugin

Damir Sudar
Hi Chris, Mark, and Nico,

Thanks you all for doing this. I will try it out later today on the nightly build of 1.4.23 and will report back.

- Damir

On 2/4/2016 1:53 PM, Chris Weisiger wrote:


On Wed, Feb 3, 2016 at 9:26 PM, Nico Stuurman <[hidden email]> wrote:

On 2/3/16 1:45 PM, Mark Tsuchida wrote:
Hi Damir,

On Wed, Feb 3, 2016 at 11:15 AM, Damir Sudar <[hidden email]> wrote:
Looks like recently there are quite a few questions about the HCS Site Generator. We actually use that extensively as well and I also have a question: I'm upgrading to 1.4.22 and noticed that you had changed the acquisition pathing from the original typewriter style to the snakelike style. That is probably great for most folks but alas for us, that is actually quite detrimental. Is there an easy way for me to change that back to typewriter style?

For background: we print a high density microarray pattern of microenvironment spots (collagen with different microenvironment proteins) and we use the HCS plugin to map that array of locations in order to image the spots in the same sequence as they are printed. So the mapping is easy. With the snakestyle, that mapping becomes suddenly difficult and very likely to get messed up.

Thanks for the suggestion. This sounds reasonable and I created a ticket for it.

Since I created the snake pattern I felt responsible and added an option to not use the snake pattern.  Regretfully, the UI code of the plugin is kind of a mess and I had to rework quite a bit to bring some sanity to it.  Since I do not have much time, problems may have slipped in, so please test at your earliest convenience and let me know of problems.  The change should be in the latest nightly build (may need to be back-ported into 2.0).

This has now been done for 2.0 as well.

-Chris


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Lawrence Berkeley Laboratory / Life Sciences Division
One Cyclotron Road, MS 977, Berkeley, CA 94720, USA
T: 510/486-5346 - F: 510/486-5586 - E: [hidden email]
WWW: http://www.lbl.gov/lifesciences/labs/sudar_lab.html

Visiting Scientist, Oregon Health & Science University

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Re: HCS plugin

PhilippeP
In reply to this post by PEARSON Matthew
PEARSON Matthew wrote
> 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?

Hi Matt,

First, sorry for the (very very) late answer. I did not see your post
earlier...
Better late than never, right?... Hmmmm.

We happily use HCS with Zeiss DF. In brief:
1- Perform plate calibration;
2- Use 3-point-AF, by going to the 3 angles of the largest virtual triangle
that would encompass the different wells you want to acquire and focusing on
your cells. Add these 3 points in a list (via "+ Mark point" button), you
can then "Set 3-point list".
You will see your chosen triangle appear on your virtual plate. You can now
delete the list, and create the real list with you grids. You will notice
that each position created has its own z value, computed from the plane
defined with your three point list.
Even if focusing is not perfect on all points when doing "Go to" in the
list, you are usually well within the working range of the Definite Focus.
Perform your acquisition with DF selected in MDA, and your pictures should
be fine. Of course, you must first set your DF up!

Hope that helps,
Philippe

PS:
While I am at it, there is still no plan on having an option to create grids
in circular pattern so as not to "waste" cells that are not present in a
square grid? Sorry to just ask without proposing to do it. I wish I could.
But my coding abilities are close to null...





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