Locations in MultiTime macro

[Gareth Howell]

Hi
I have two questions relating to the MT macro which I'm using for screening
96 well plates. Currently I manually set the positions relating to each
plate and save them within the macro using the Store/Apply step. However
when they are recovered some (~10%) are missing the wells and we loose data
within the screens. So my questions are:
1) is it possible to edit the locations within the Store/Apply so that they
are all accurate?
2) is it possible to save each co-ordinate as a seperate configuration, with
the stage coordinates included, and run the screen as a series of blocks
within the macro? I realise this would be initially very labour intensive
but worth the effort in the long run.
Thanks
Gareth

[Danielle Crippen]

Dear Gareth,

First of all, I'd recommend calling Zeiss and talking with Zbi, Maya or John about this.  You can get advice from them regardless of whether you're on service contract...call 1-800-633-6610.  I find them to be endless resources.  (BTW--I have no affiliation with Zeiss...just use their equipment a lot).

Secondly...I'm wondering...are you zero-ing the stage?  Do you have a stage with linear encoders on it for the best accuracy?  How are you ensuring you're re-placing your plate in the exact same location as when you originally marked positions.  If you're hoping to image the same ROI (with the same cells in it etc), even a 50-100um xy shift can ruin your coordinates.  We've had some good success here with 8 well chambered coverglass by zeroing the stage at a particular corner of a particular well.  Then when we re-load the chamber, we zero again with the same method...then we can successfully return to previously imaged coordinates for further imaging...even weeks later.  

I think in answer to your first question, you can of course always manually re-do locations by moving the stage to the well you want to image, selecting that location in the MT list, and clicking the "replace xyz" button.  Once you've corrected each location, you can Store/Apply again and overwrite the MT setup file.

I can't comment on your second question, b/c I don't utilize the blocks in MT (yet:)...so I'm not sure how or if this would work.

Best of luck!!

d


Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Age Research
8001 Redwood Blvd
Novato, CA 94945
415-209-2046



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gareth Howell
Sent: Friday, June 19, 2009 4:31 AM
To: [hidden email]
Subject: Locations in MultiTime macro

Hi
I have two questions relating to the MT macro which I'm using for screening
96 well plates. Currently I manually set the positions relating to each plate and save them within the macro using the Store/Apply step. However when they are recovered some (~10%) are missing the wells and we loose data within the screens. So my questions are:
1) is it possible to edit the locations within the Store/Apply so that they are all accurate?
2) is it possible to save each co-ordinate as a seperate configuration, with the stage coordinates included, and run the screen as a series of blocks within the macro? I realise this would be initially very labour intensive but worth the effort in the long run.
Thanks
Gareth

[Armstrong, Brian]

Hi Gareth, with regards to question 2, I think you MUST store the
position as a separate configuration in order for it to work properly.
This will have your x,y, and z positions stored.
 
If you do this your first question will be unnecassary.
Cheers,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Danielle Crippen
Sent: Friday, June 19, 2009 9:08 AM
To: [hidden email]
Subject: Re: Locations in MultiTime macro

Dear Gareth,

First of all, I'd recommend calling Zeiss and talking with Zbi, Maya or
John about this.  You can get advice from them regardless of whether
you're on service contract...call 1-800-633-6610.  I find them to be
endless resources.  (BTW--I have no affiliation with Zeiss...just use
their equipment a lot).

Secondly...I'm wondering...are you zero-ing the stage?  Do you have a
stage with linear encoders on it for the best accuracy?  How are you
ensuring you're re-placing your plate in the exact same location as when
you originally marked positions.  If you're hoping to image the same ROI
(with the same cells in it etc), even a 50-100um xy shift can ruin your
coordinates.  We've had some good success here with 8 well chambered
coverglass by zeroing the stage at a particular corner of a particular
well.  Then when we re-load the chamber, we zero again with the same
method...then we can successfully return to previously imaged
coordinates for further imaging...even weeks later.  

I think in answer to your first question, you can of course always
manually re-do locations by moving the stage to the well you want to
image, selecting that location in the MT list, and clicking the "replace
xyz" button.  Once you've corrected each location, you can Store/Apply
again and overwrite the MT setup file.

I can't comment on your second question, b/c I don't utilize the blocks
in MT (yet:)...so I'm not sure how or if this would work.

Best of luck!!

d


Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Age Research
8001 Redwood Blvd
Novato, CA 94945
415-209-2046



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Gareth Howell
Sent: Friday, June 19, 2009 4:31 AM
To: [hidden email]
Subject: Locations in MultiTime macro

Hi
I have two questions relating to the MT macro which I'm using for
screening
96 well plates. Currently I manually set the positions relating to each
plate and save them within the macro using the Store/Apply step. However
when they are recovered some (~10%) are missing the wells and we loose
data within the screens. So my questions are:
1) is it possible to edit the locations within the Store/Apply so that
they are all accurate?
2) is it possible to save each co-ordinate as a seperate configuration,
with the stage coordinates included, and run the screen as a series of
blocks within the macro? I realise this would be initially very labour
intensive but worth the effort in the long run.
Thanks
Gareth


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[cromey]

Regarding the resetting of a zero position: On a different instrument where
we did automated analysis of 96 well plates, we created a fiduciary mark on
a blank plate and set that as zero every time we ran the analysis.  All the
measurement positions were created from that initial point.  To make the
mark we scratched a cross-hair on the plate's bottom surface using a sharp
razor blade.

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Assistant Scientific Investigator
Dept. of Cell Biology & Anatomy, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/exppath/
Home of: "Microscopy and Imaging Resources on the WWW"


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Danielle Crippen
Sent: Friday, June 19, 2009 9:08 AM
To: [hidden email]
Subject: Re: Locations in MultiTime macro

Dear Gareth,

First of all, I'd recommend calling Zeiss and talking with Zbi, Maya or John
about this.  You can get advice from them regardless of whether you're on
service contract...call 1-800-633-6610.  I find them to be endless
resources.  (BTW--I have no affiliation with Zeiss...just use their
equipment a lot).

Secondly...I'm wondering...are you zero-ing the stage?  Do you have a stage
with linear encoders on it for the best accuracy?  How are you ensuring
you're re-placing your plate in the exact same location as when you
originally marked positions.  If you're hoping to image the same ROI (with
the same cells in it etc), even a 50-100um xy shift can ruin your
coordinates.  We've had some good success here with 8 well chambered
coverglass by zeroing the stage at a particular corner of a particular well.
Then when we re-load the chamber, we zero again with the same method...then
we can successfully return to previously imaged coordinates for further
imaging...even weeks later.  

I think in answer to your first question, you can of course always manually
re-do locations by moving the stage to the well you want to image, selecting
that location in the MT list, and clicking the "replace xyz" button.  Once
you've corrected each location, you can Store/Apply again and overwrite the
MT setup file.

I can't comment on your second question, b/c I don't utilize the blocks in
MT (yet:)...so I'm not sure how or if this would work.

Best of luck!!

d


Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Age Research
8001 Redwood Blvd
Novato, CA 94945
415-209-2046



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Gareth Howell
Sent: Friday, June 19, 2009 4:31 AM
To: [hidden email]
Subject: Locations in MultiTime macro

Hi
I have two questions relating to the MT macro which I'm using for screening
96 well plates. Currently I manually set the positions relating to each
plate and save them within the macro using the Store/Apply step. However
when they are recovered some (~10%) are missing the wells and we loose data
within the screens. So my questions are:
1) is it possible to edit the locations within the Store/Apply so that they
are all accurate?
2) is it possible to save each co-ordinate as a seperate configuration, with
the stage coordinates included, and run the screen as a series of blocks
within the macro? I realise this would be initially very labour intensive
but worth the effort in the long run.
Thanks
Gareth

[Gert van Cappellen]

I don't think option 2 is going to work, because the configurations do
not store the stage position. The Zeiss multi-time macro has however an
option  to define a grid under the button edit locations. You can define
this grid with the dimensions of your 96 well plate. So you focus on the
well with the lowest X and Y and than you define the grid. Probably the
first time you have to check the dimensions and probably fine tune this
a bit. The settings will be remembered so the next time pushing one
button will do.

Good luck,
Gert van Cappellen

on 19-6-2009 13:31 Gareth Howell said the following:

> Hi
> I have two questions relating to the MT macro which I'm using for screening
> 96 well plates. Currently I manually set the positions relating to each
> plate and save them within the macro using the Store/Apply step. However
> when they are recovered some (~10%) are missing the wells and we loose data
> within the screens. So my questions are:
> 1) is it possible to edit the locations within the Store/Apply so that they
> are all accurate?
> 2) is it possible to save each co-ordinate as a seperate configuration, with
> the stage coordinates included, and run the screen as a series of blocks
> within the macro? I realise this would be initially very labour intensive
> but worth the effort in the long run.
> Thanks
> Gareth
>  

--
Gert van Cappellen
TEL [phone]; FAX [phone]; Email [hidden email]
Optical Imaging Centre (OIC); http://www.erasmusmc.nl/oic/
Dept. of Reproduction and Development; http://www.erasmusmc.nl/rede
Room Ee914, Erasmus MC, Dr. Molenwaterplein 50, 3015 GE ROTTERDAM, The Netherlands