Pinhole adjustment Zeiss LSM 510

[Jolien Tyler-2]

*****
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Hello All,

I would like to use PHAdjust to adjust pinholes, and have run into a
problem.  From a previous post on this listserv (
http://lists.umn.edu/cgi-bin/wa?A2=ind0902&L=confocalmicroscopy&P=25435) -

"PHAdjust adjusts only single tracks for the logged-in user. Thus we have
one account where we collect all potential configurations and use this
log-in to align the pinholes"

Since we have unique logins for each user, how do I collect all potential
configurations so that I only have to perform PHAdjust for a single
Administrator user?  Are the pinhole adjustments done for that user "saved"
to all user accounts?  Any advice for maintaining pinholes on the Zeiss LSM
510 with multiple user accounts?

Thank you,
Jolien


-----------------------------------------------
Jolien Tyler, Ph.D.

University of Colorado
MCD Biology
347 UCB
Boulder, CO 80309
303-492-5955 (office)

[Keith Morris]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Jolien,

Just noticed your Zeiss 510 PHAdjust post from last month and can't see any replies. Ironically we have just gone to a single account login as our Zeiss 510 PC has had to come off the building network as LMS510 runs under Windows XP.

Prior to that though we had the same issue, as we all logged in separately via our WTCHG network accounts and may have had a few different non-standard Zeiss 510 confocal filter configs each. All you need to do is log-in as yourself onto the 510 imaging PC (assuming you have admin rights) and copy and paste all the relevant Zeiss 510 lsm images (created with unusual configs) in other user Zeiss 510 databases into a new Zeiss image mdb database you have created (using the copy and paste buttons on the LMS510 database, ie. via the 510 database window with the form, gallery, & table options). Once all the 510 lsm images captured using configs that need pinhole adjustment are in your personal 510 mdb database,  it's then a simple matter to click on the reuse button to load the images filter configs and settings into your LMS510 software and save each of the unusual filter config combos individually (any name will do). Not thought about it before, but it's only the unique combination of filters that need to be presented to  PHAdjust not the specific name the user gave to that filter combo. In practice you should only need to do this with any users that get the 'Pinhole not adjusted' warning when they try to scan, as they are the ones with a non-standard filter combo that hasn't been auto-aligned by PHAdjust before.

Most users will have exactly the same standard Zeiss config combo of filters (e.g. DAPI, FITC, TRICT, Cy5) despite the different names they gave them, so they will most likely be done with either the standard Zeiss configs or those you created yourself with your account for your specimens. PhAdjust only works on configs stored with the LMS510 user account that is logged in so you do have to manually add in any new 'pinhole unadjusted' filter configs that you don't have in your account (which in practice probably won't be many, e.g. perhaps TexasRed YFP, CFP etc.). Hope this helps. Winfried was saying the same thing in his 2009 post, but I've just expanded it. If your users have removed all the images from the Zeiss PC that have the 'pinhole not adjusted' configs they will either have to create a new image for you or find an old database image taken with those settings to pass on.

PS. There is a way for Ziess engineers to backup all the user filter configs from all user logins into one account (say if they are replacing PCs) but it stores all of the user configs in one long list even if the majority are identical and you get a bit overwhelmed with repetitions (which PHAdjust will ignore though, as it will only pinhole align one specific filter combo once) - to keep it simple I prefer the above copy/paste method.  Any problems feel free to contact me off list.

Regards

Keith

-----------------------------------------------------------
Dr Keith J Morris
Cellular Imaging Microscopy Core,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford,
OX3 7BN,
United Kingdom.
 
Tel:  +44   ( 0 ) 1865  287568
Email:   [hidden email]
Webpage: www.well.ox.ac.uk/microscopy-facilities
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jolien Tyler
Sent: 18 June 2014 21:30
To: [hidden email]
Subject: Pinhole adjustment Zeiss LSM 510

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello All,

I would like to use PHAdjust to adjust pinholes, and have run into a problem.  From a previous post on this listserv (
http://lists.umn.edu/cgi-bin/wa?A2=ind0902&L=confocalmicroscopy&P=25435) -

"PHAdjust adjusts only single tracks for the logged-in user. Thus we have one account where we collect all potential configurations and use this log-in to align the pinholes"

Since we have unique logins for each user, how do I collect all potential configurations so that I only have to perform PHAdjust for a single Administrator user?  Are the pinhole adjustments done for that user "saved"
to all user accounts?  Any advice for maintaining pinholes on the Zeiss LSM
510 with multiple user accounts?

Thank you,
Jolien


-----------------------------------------------
Jolien Tyler, Ph.D.

University of Colorado
MCD Biology
347 UCB
Boulder, CO 80309
303-492-5955 (office)

[Jolien Tyler-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello All,
Thanks for the detailed responses.  I did also receive a detailed response
from the folks at Zeiss off list, which confirmed that the pinhole
adjustment depends on the combinations of dichroics leading to a particular
channel.  I have thus discovered that the vast majority of users are
unaffected by unique combos that are not already present.  Thanks Keith for
pointing out the quick way to get those configs into a single user account
- should anyone get very creative I'll use that approach.  For anyone else
who may run into this issue, the pinhole adjustments do carry over to all
existing users (unlike the filter configs).  I ran it with 10x objective,
633 nm laser line, and a regular glass slide without any problems.
Thanks,
Jolien


-----------------------------------------------
Jolien Tyler, Ph.D.

University of Colorado
MCD Biology
347 UCB
Boulder, CO 80309
303-492-5955 (office)


On Wed, Jul 2, 2014 at 6:52 AM, Keith Morris <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Jolien,
>
> Just noticed your Zeiss 510 PHAdjust post from last month and can't see
> any replies. Ironically we have just gone to a single account login as our
> Zeiss 510 PC has had to come off the building network as LMS510 runs under
> Windows XP.
>
> Prior to that though we had the same issue, as we all logged in separately
> via our WTCHG network accounts and may have had a few different
> non-standard Zeiss 510 confocal filter configs each. All you need to do is
> log-in as yourself onto the 510 imaging PC (assuming you have admin rights)
> and copy and paste all the relevant Zeiss 510 lsm images (created with
> unusual configs) in other user Zeiss 510 databases into a new Zeiss image
> mdb database you have created (using the copy and paste buttons on the
> LMS510 database, ie. via the 510 database window with the form, gallery, &
> table options). Once all the 510 lsm images captured using configs that
> need pinhole adjustment are in your personal 510 mdb database,  it's then a
> simple matter to click on the reuse button to load the images filter
> configs and settings into your LMS510 software and save each of the unusual
> filter config combos individually (any name will do). Not thought about it
> before, but it's only the unique combination of filters that need to be
> presented to  PHAdjust not the specific name the user gave to that filter
> combo. In practice you should only need to do this with any users that get
> the 'Pinhole not adjusted' warning when they try to scan, as they are the
> ones with a non-standard filter combo that hasn't been auto-aligned by
> PHAdjust before.
>
> Most users will have exactly the same standard Zeiss config combo of
> filters (e.g. DAPI, FITC, TRICT, Cy5) despite the different names they gave
> them, so they will most likely be done with either the standard Zeiss
> configs or those you created yourself with your account for your specimens.
> PhAdjust only works on configs stored with the LMS510 user account that is
> logged in so you do have to manually add in any new 'pinhole unadjusted'
> filter configs that you don't have in your account (which in practice
> probably won't be many, e.g. perhaps TexasRed YFP, CFP etc.). Hope this
> helps. Winfried was saying the same thing in his 2009 post, but I've just
> expanded it. If your users have removed all the images from the Zeiss PC
> that have the 'pinhole not adjusted' configs they will either have to
> create a new image for you or find an old database image taken with those
> settings to pass on.
>
> PS. There is a way for Ziess engineers to backup all the user filter
> configs from all user logins into one account (say if they are replacing
> PCs) but it stores all of the user configs in one long list even if the
> majority are identical and you get a bit overwhelmed with repetitions
> (which PHAdjust will ignore though, as it will only pinhole align one
> specific filter combo once) - to keep it simple I prefer the above
> copy/paste method.  Any problems feel free to contact me off list.
>
> Regards
>
> Keith
>
> -----------------------------------------------------------
> Dr Keith J Morris
> Cellular Imaging Microscopy Core,
> The Wellcome Trust Centre for Human Genetics,
> Roosevelt Drive,
> Oxford,
> OX3 7BN,
> United Kingdom.
>
> Tel:  +44   ( 0 ) 1865  287568
> Email:   [hidden email]
> Webpage: www.well.ox.ac.uk/microscopy-facilities
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Jolien Tyler
> Sent: 18 June 2014 21:30
> To: [hidden email]
> Subject: Pinhole adjustment Zeiss LSM 510
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello All,
>
> I would like to use PHAdjust to adjust pinholes, and have run into a
> problem.  From a previous post on this listserv (
> http://lists.umn.edu/cgi-bin/wa?A2=ind0902&L=confocalmicroscopy&P=25435) -
>
> "PHAdjust adjusts only single tracks for the logged-in user. Thus we have
> one account where we collect all potential configurations and use this
> log-in to align the pinholes"
>
> Since we have unique logins for each user, how do I collect all potential
> configurations so that I only have to perform PHAdjust for a single
> Administrator user?  Are the pinhole adjustments done for that user "saved"
> to all user accounts?  Any advice for maintaining pinholes on the Zeiss LSM
> 510 with multiple user accounts?
>
> Thank you,
> Jolien
>
>
> -----------------------------------------------
> Jolien Tyler, Ph.D.
>
> University of Colorado
> MCD Biology
> 347 UCB
> Boulder, CO 80309
> 303-492-5955 (office)
>

[Keith Morris]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Glad you got it sorted Jolien.

Zeiss full maintenance support for the Zeiss 510 confocal series stops in mid 2016 after which Zeiss tell me there won't be any formal annual maintenance contracts available, and parts/repairs after that will be on as is basis.

As you say running PHAdusts aligns the pinholes for all users that have that filter configuration. Just out of interest when users click on the re-use button on an old lsm image, the reused filter combinations does reset the pinhole diameter to that used to capture the old image but it doesn’t change the pinhole alignment to the original one (it keeps the pinhole alignment at the latest pinhole X,Y settings). It does however reset the collimator to the original images setting, which can affect evenness of the illumination across the field (for us that's the blue DAPI channel as our scanhead has gone little out of alignment over the years). If you get fed up with a 'pinhole not aligned' error message you can go into Maintain, Pinhole and just click save, reload for the PMT channel affected, and for us that prevents the annoying error message coming up again (you can run PHAdjust later to set up the pinhole correctly for that new filter combo - PHAdjust is always carried out with the 10x objective and 633nm laser after leaving it warming up for a few hours, and I use the Zeiss mirror slide as I still have it lying about).

Regards

Keith

-----------------------------------------------------------
Dr Keith J Morris
Cellular Imaging Microscopy Core,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford,
OX3 7BN,
United Kingdom.
 
Tel:  +44   ( 0 ) 1865  287568
Email:   [hidden email]
Webpage: www.well.ox.ac.uk/microscopy-facilities
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jolien Tyler
Sent: 02 July 2014 17:47
To: [hidden email]
Subject: Re: Pinhole adjustment Zeiss LSM 510

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello All,
Thanks for the detailed responses.  I did also receive a detailed response from the folks at Zeiss off list, which confirmed that the pinhole adjustment depends on the combinations of dichroics leading to a particular channel.  I have thus discovered that the vast majority of users are unaffected by unique combos that are not already present.  Thanks Keith for pointing out the quick way to get those configs into a single user account
- should anyone get very creative I'll use that approach.  For anyone else who may run into this issue, the pinhole adjustments do carry over to all existing users (unlike the filter configs).  I ran it with 10x objective,
633 nm laser line, and a regular glass slide without any problems.
Thanks,
Jolien


-----------------------------------------------
Jolien Tyler, Ph.D.

University of Colorado
MCD Biology
347 UCB
Boulder, CO 80309
303-492-5955 (office)


On Wed, Jul 2, 2014 at 6:52 AM, Keith Morris <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Jolien,
>
> Just noticed your Zeiss 510 PHAdjust post from last month and can't
> see any replies. Ironically we have just gone to a single account
> login as our Zeiss 510 PC has had to come off the building network as
> LMS510 runs under Windows XP.
>
> Prior to that though we had the same issue, as we all logged in
> separately via our WTCHG network accounts and may have had a few
> different non-standard Zeiss 510 confocal filter configs each. All you
> need to do is log-in as yourself onto the 510 imaging PC (assuming you
> have admin rights) and copy and paste all the relevant Zeiss 510 lsm
> images (created with unusual configs) in other user Zeiss 510
> databases into a new Zeiss image mdb database you have created (using
> the copy and paste buttons on the
> LMS510 database, ie. via the 510 database window with the form,
> gallery, & table options). Once all the 510 lsm images captured using
> configs that need pinhole adjustment are in your personal 510 mdb
> database,  it's then a simple matter to click on the reuse button to
> load the images filter configs and settings into your LMS510 software
> and save each of the unusual filter config combos individually (any
> name will do). Not thought about it before, but it's only the unique
> combination of filters that need to be presented to  PHAdjust not the
> specific name the user gave to that filter combo. In practice you
> should only need to do this with any users that get the 'Pinhole not
> adjusted' warning when they try to scan, as they are the ones with a
> non-standard filter combo that hasn't been auto-aligned by PHAdjust before.
>
> Most users will have exactly the same standard Zeiss config combo of
> filters (e.g. DAPI, FITC, TRICT, Cy5) despite the different names they
> gave them, so they will most likely be done with either the standard
> Zeiss configs or those you created yourself with your account for your specimens.
> PhAdjust only works on configs stored with the LMS510 user account
> that is logged in so you do have to manually add in any new 'pinhole unadjusted'
> filter configs that you don't have in your account (which in practice
> probably won't be many, e.g. perhaps TexasRed YFP, CFP etc.). Hope
> this helps. Winfried was saying the same thing in his 2009 post, but
> I've just expanded it. If your users have removed all the images from
> the Zeiss PC that have the 'pinhole not adjusted' configs they will
> either have to create a new image for you or find an old database
> image taken with those settings to pass on.
>
> PS. There is a way for Ziess engineers to backup all the user filter
> configs from all user logins into one account (say if they are
> replacing
> PCs) but it stores all of the user configs in one long list even if
> the majority are identical and you get a bit overwhelmed with
> repetitions (which PHAdjust will ignore though, as it will only
> pinhole align one specific filter combo once) - to keep it simple I
> prefer the above copy/paste method.  Any problems feel free to contact me off list.
>
> Regards
>
> Keith
>
> -----------------------------------------------------------
> Dr Keith J Morris
> Cellular Imaging Microscopy Core,
> The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford,
> OX3 7BN,
> United Kingdom.
>
> Tel:  +44   ( 0 ) 1865  287568
> Email:   [hidden email]
> Webpage: www.well.ox.ac.uk/microscopy-facilities
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Jolien Tyler
> Sent: 18 June 2014 21:30
> To: [hidden email]
> Subject: Pinhole adjustment Zeiss LSM 510
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello All,
>
> I would like to use PHAdjust to adjust pinholes, and have run into a
> problem.  From a previous post on this listserv (
> http://lists.umn.edu/cgi-bin/wa?A2=ind0902&L=confocalmicroscopy&P=2543
> 5) -
>
> "PHAdjust adjusts only single tracks for the logged-in user. Thus we
> have one account where we collect all potential configurations and use
> this log-in to align the pinholes"
>
> Since we have unique logins for each user, how do I collect all
> potential configurations so that I only have to perform PHAdjust for a
> single Administrator user?  Are the pinhole adjustments done for that user "saved"
> to all user accounts?  Any advice for maintaining pinholes on the
> Zeiss LSM
> 510 with multiple user accounts?
>
> Thank you,
> Jolien
>
>
> -----------------------------------------------
> Jolien Tyler, Ph.D.
>
> University of Colorado
> MCD Biology
> 347 UCB
> Boulder, CO 80309
> 303-492-5955 (office)
>