uniform focus across the field question

[Cammer, Michael-2]

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Dear confocalers and other light microscopists,

We have a question about tilt of a specimen or detectors leading to shift of focus across a field.

From the first confocal microscope we used in 1991 through many other microscopes over the years, we have not had problems with planapochromat lenses with the image being in focus corner to corner across the field.  This includes newer microscopes with wider fields of view and a spinning disk confocal with a Cairn twin camera setup.  (The cameras, however, required substantial alignment to attain this, but it has been locked in place for over a year.)  We are used to putting in standard stage inserts or even unusual ones, a variety of samples, and being able to focus at the substrate across the entire field.  This includes imaging in reflection mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent confocal.  This is routine.

We have a new microscope, however, which does not have this focus uniformity.  I believe it is a dual camera alignment issue and we have been discussing this for almost a year as an installation issue, but recently the manufacturer insists that the only way to fix the problem we need to use screws on the stage to adjust the tilt.  I am concerned that this is going to be a major problem in a core facility with users who will play with anything.  It is going to add substantial burden on staff.  Also, it means that standard inserts will not work.  And it makes me wonder what this will do to the PSF if the coverglass isn't perpendicular to the light path.

Have we been unusually lucky all these years to never have encountered this problem before?  Are we unreasonable to expect the microscope to be installed with all planes parallel for routine use?  Is this type of stage alignment standard acceptable procedure in other labs?

Thank you.



Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

[hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/

Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567


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[George McNamara]

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Hi Michael,

There is no tolerance on how level a microscope stage, or its specimen
holding insert (ex. K-Frame) is supposed to be for research microscopes.
In fact, other than #1.5 coverglasses being ~170 um of glass made of
[whatever] (ok, and similar specifications for #0, #1, #2, etc), there
are not a lot of specifications (and most of the specifications, re:
ISO, are just to try to cause innovators to be slowed down by writing
mountains of documentation).

I suggest: get an "optical flat" (my local Olympus rep had one ... or
maybe it floats with some of O's demo equipment ... if too expensive,
then a plain glass slide, image, turn around 180 degrees, image, if
consistent, hope it is "flat" enough), and use reflected light -- for a
widefield filter set to do this see, for example, my
http://confocal.jhu.edu/current-equipment/fishscope page (RT10/90 or
RT03/97 -- either one is fine for this, and for interference reflection
light microscope of cell-substratum interactions ... many confocals have
R10/T90 or similar master[mistress?] beamsplitter) -- to determine if
the two cameras simultaneously see the same thing or not (flat or not).
If not, then use the "stage screws" (i.e. the screws on the corners of a
K-frame insert) to level the insert with respect to one or hopefully
both. If only one is flat, follow whatever instructions the dual camera
thingy manufacturer recommends to get the other to match.

This becomes the "ground truth" and users are then responsible for doing
their best to make their coverglass-slide and imaging dish purchases and
"assemblies" (ex: no label or coverglass at one edge of a slide) be flat
with respect to your "truth".

Document all your steps ... including  (hopefully) that that your
"optical flat" remains constant over days, weeks, months, then publish
one or more articles (or preprints, then articles, then protocol articles).

enjoy,

George

p.s. except for TIRF filter cubes (and maybe not even then) the
fluorescence filter companies (i.e. Chroma, Semrock) do not claim their
glass is flat (or exactly 45 degrees for beamsplitters ... hmmm ... some
companies may deliberately tilt their emission filters from being
exactly perpendicular to the light path), so different filter cubes
could impact the microscope flatness ... thin layer of fluorescent stuff
(or small beads) or Michael Model's approach of high concentration
fluorescent dyes could be useful.

p.p.s. "you have another filter(s)" - whatever is splitting the light
path between the two cameras may not have perfectly "optical flat"
glasses. Maybe that component should have a TIRF quality beamsplitter
and high quality emission filters.

p.p.s. having (or buying on amazon.com, Home Depot, etc) a "bubble
level" could tell you if your microscope stage is level wrt gravity. I
suggest no need for something like "BLACK+DECKER Laser Level (BDL220S)"
(amazon.com product, currently $14.52) ... if you do buy a laser
leveler, please keep in mind: "do not look at laser with remaining eye".


On 6/15/2021 9:21 PM, Cammer, Michael 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.
> *****
>
> Dear confocalers and other light microscopists,
>
> We have a question about tilt of a specimen or detectors leading to shift of focus across a field.
>
>  From the first confocal microscope we used in 1991 through many other microscopes over the years, we have not had problems with planapochromat lenses with the image being in focus corner to corner across the field.  This includes newer microscopes with wider fields of view and a spinning disk confocal with a Cairn twin camera setup.  (The cameras, however, required substantial alignment to attain this, but it has been locked in place for over a year.)  We are used to putting in standard stage inserts or even unusual ones, a variety of samples, and being able to focus at the substrate across the entire field.  This includes imaging in reflection mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent confocal.  This is routine.
>
> We have a new microscope, however, which does not have this focus uniformity.  I believe it is a dual camera alignment issue and we have been discussing this for almost a year as an installation issue, but recently the manufacturer insists that the only way to fix the problem we need to use screws on the stage to adjust the tilt.  I am concerned that this is going to be a major problem in a core facility with users who will play with anything.  It is going to add substantial burden on staff.  Also, it means that standard inserts will not work.  And it makes me wonder what this will do to the PSF if the coverglass isn't perpendicular to the light path.
>
> Have we been unusually lucky all these years to never have encountered this problem before?  Are we unreasonable to expect the microscope to be installed with all planes parallel for routine use?  Is this type of stage alignment standard acceptable procedure in other labs?
>
> Thank you.
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
>
> [hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
> ------------------------------------------------------------
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> =================================

[Dan Focht]

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Michael


As a former Zeiss affiliate service tech, back when I was young, and this still applies today, we used a stage square to make sure that the flat surface of the nosepiece and the surface of the stage were parallel with each other. The device was made on a lathe. The base was about 24mm diameter and screwed into the nosepiece and extended 45mm to a flat disk about 75mm round. You would carefully lower the 75mm disk to the stage surface to make sure it touched evenly. Can be used on uprights or inverts.
This enabled the user to check for parallelism issues caused by either the stage mount or the detent in the nosepiece. If the detent wore it caused a rotational misalignment which causes the focus to be nonuniform across the field. It is also best to have a crosshair objective to check alignment of the optical axis of the scope. Check with your scope manufacturer to see if they can or will provide these reference parts.



Dan Focht
[hidden email]

> On Jun 15, 2021, at 9:21 PM, Cammer, Michael <[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.
> *****
>
> Dear confocalers and other light microscopists,
>
> We have a question about tilt of a specimen or detectors leading to shift of focus across a field.
>
> From the first confocal microscope we used in 1991 through many other microscopes over the years, we have not had problems with planapochromat lenses with the image being in focus corner to corner across the field.  This includes newer microscopes with wider fields of view and a spinning disk confocal with a Cairn twin camera setup.  (The cameras, however, required substantial alignment to attain this, but it has been locked in place for over a year.)  We are used to putting in standard stage inserts or even unusual ones, a variety of samples, and being able to focus at the substrate across the entire field.  This includes imaging in reflection mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent confocal.  This is routine.
>
> We have a new microscope, however, which does not have this focus uniformity.  I believe it is a dual camera alignment issue and we have been discussing this for almost a year as an installation issue, but recently the manufacturer insists that the only way to fix the problem we need to use screws on the stage to adjust the tilt.  I am concerned that this is going to be a major problem in a core facility with users who will play with anything.  It is going to add substantial burden on staff.  Also, it means that standard inserts will not work.  And it makes me wonder what this will do to the PSF if the coverglass isn't perpendicular to the light path.
>
> Have we been unusually lucky all these years to never have encountered this problem before?  Are we unreasonable to expect the microscope to be installed with all planes parallel for routine use?  Is this type of stage alignment standard acceptable procedure in other labs?
>
> Thank you.
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
>
> [hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

[Jonkman, James]

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Hi, Michael.  We have a dozen confocals and none of them have any appreciable focus shift across a single field of view.  In fact, on the inverted microscopes with a thin sample if a user has a label close to one edge of the slide and no label on the other, this should cause an obvious tilt as the slide sits higher on one side than the other - but from a single field of view it isn't very obvious unless you're looking for it.  The effect of the label becomes obvious if you move the stage - the sample quickly goes out of focus as you move side to side.  If there is a substantial tilt across the field of view I would agree that the manufacturer should address this, and not with stage inserts: we usually remove the stage insert tilt screws as they tend to jiggle their way down with gravity and end up misaligning the stage tilt more than anything else.

Maybe you want to create a shared cloud folder (Dropbox, Onedrive, ...) and drop an image or 2 in there so that we can see how bad it is?  If it would help, I would be willing to take some images across several of our confocals and upload them to the same folder.  I'm thinking a short z-stack of a thin sample (Molecular Probes Prepared Slide #1, BPAE cells for example) would illustrate the problem nicely.  And maybe a second test would be to focus the same slide in the middle of the field of view, then take a tiled image (say, 5 x 5). Choose an objective and we can try to match it.  Anyone else interested?  If not I'm happy to discuss further offline.

Cheers,
James

-----------------------------------------------
   James Jonkman, Staff Scientist
   Advanced Optical Microscopy Facility (AOMF)
   and Wright Cell Imaging Facility (WCIF)
   University Health Network
   MaRS, PMCRT tower, 101 College St., Room 15-305
   Toronto, ON, CANADA    M5G 1L7
  [hidden email]  Tel: [phone]
   www.aomf.ca


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: Tuesday, June 15, 2021 9:21 PM
To: [hidden email]
Subject: [External] uniform focus across the field question

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*****

Dear confocalers and other light microscopists,

We have a question about tilt of a specimen or detectors leading to shift of focus across a field.

From the first confocal microscope we used in 1991 through many other microscopes over the years, we have not had problems with planapochromat lenses with the image being in focus corner to corner across the field.  This includes newer microscopes with wider fields of view and a spinning disk confocal with a Cairn twin camera setup.  (The cameras, however, required substantial alignment to attain this, but it has been locked in place for over a year.)  We are used to putting in standard stage inserts or even unusual ones, a variety of samples, and being able to focus at the substrate across the entire field.  This includes imaging in reflection mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent confocal.  This is routine.

We have a new microscope, however, which does not have this focus uniformity.  I believe it is a dual camera alignment issue and we have been discussing this for almost a year as an installation issue, but recently the manufacturer insists that the only way to fix the problem we need to use screws on the stage to adjust the tilt.  I am concerned that this is going to be a major problem in a core facility with users who will play with anything.  It is going to add substantial burden on staff.  Also, it means that standard inserts will not work.  And it makes me wonder what this will do to the PSF if the coverglass isn't perpendicular to the light path.

Have we been unusually lucky all these years to never have encountered this problem before?  Are we unreasonable to expect the microscope to be installed with all planes parallel for routine use?  Is this type of stage alignment standard acceptable procedure in other labs?

Thank you.



Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

[hidden email]<mailto:[hidden email]>  https://urldefense.com/v3/__http://nyulmc.org/micros__;!!CjcC7IQ!Z7U94tM1sMtYZhhlvDZ6lwfd94ioWz9rYEzmfx_koBo2Yd27Vv0vHrKYYJniWk7qagUc$ [nyulmc[.]org]  https://urldefense.com/v3/__http://microscopynotes.com/__;!!CjcC7IQ!Z7U94tM1sMtYZhhlvDZ6lwfd94ioWz9rYEzmfx_koBo2Yd27Vv0vHrKYYJniWvaaQUMl$ [microscopynotes[.]com]

Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567


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[Douglas Richardson]

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Hi Michael,

As a follow up to our previous conversation:

This microscope (others can see the previous string, it's not a confocal)
comes standard with a unique levelling stage insert. It has X and Y
adjustment screws in opposite corners and a pivot point in the other
corner. This stage insert can correct for any tilt throughout the system,
although it can take a long time to get it right.

Since you mentioned TIRF, I assume you're not using that insert, but a live
cell heated stage insert? I do not like the standard heated stage that
ships with that system. In our facility it eats objectives. Therefore, we
ordered the Pecon push and click system for live cell holders (and rely on
the plastic incubation box + time to maintain temperature). We had to sign
a waiver acknowledging that this insert was not approved for use with the
system and that we may experience unwanted thermal drift and uncorrectable
tilt.

It turns out we didn't see any of this and now exclusively use the push and
click system for live and fixed samples as it can also hold slides. The
adjustable stage now collects dust in a box and our images look great. No
need for adjusting stage screws.

To be fair, we don't do TIRF, but we image 200 um beads at every SIM
session and these are always in focus across the field. It was very
difficult to achieve similar uniformity with the adjustable stage unless
you put in a good 30-45 mins of adjustments.

-Doug

On Wed, Jun 16, 2021 at 9:16 AM Jonkman, James <[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, Michael.  We have a dozen confocals and none of them have any
> appreciable focus shift across a single field of view.  In fact, on the
> inverted microscopes with a thin sample if a user has a label close to one
> edge of the slide and no label on the other, this should cause an obvious
> tilt as the slide sits higher on one side than the other - but from a
> single field of view it isn't very obvious unless you're looking for it.
> The effect of the label becomes obvious if you move the stage - the sample
> quickly goes out of focus as you move side to side.  If there is a
> substantial tilt across the field of view I would agree that the
> manufacturer should address this, and not with stage inserts: we usually
> remove the stage insert tilt screws as they tend to jiggle their way down
> with gravity and end up misaligning the stage tilt more than anything else.
>
> Maybe you want to create a shared cloud folder (Dropbox, Onedrive, ...)
> and drop an image or 2 in there so that we can see how bad it is?  If it
> would help, I would be willing to take some images across several of our
> confocals and upload them to the same folder.  I'm thinking a short z-stack
> of a thin sample (Molecular Probes Prepared Slide #1, BPAE cells for
> example) would illustrate the problem nicely.  And maybe a second test
> would be to focus the same slide in the middle of the field of view, then
> take a tiled image (say, 5 x 5). Choose an objective and we can try to
> match it.  Anyone else interested?  If not I'm happy to discuss further
> offline.
>
> Cheers,
> James
>
> -----------------------------------------------
>    James Jonkman, Staff Scientist
>    Advanced Optical Microscopy Facility (AOMF)
>    and Wright Cell Imaging Facility (WCIF)
>    University Health Network
>    MaRS, PMCRT tower, 101 College St., Room 15-305
>    Toronto, ON, CANADA    M5G 1L7
>   [hidden email]  Tel: [phone]
>    www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Cammer, Michael
> Sent: Tuesday, June 15, 2021 9:21 PM
> To: [hidden email]
> Subject: [External] uniform focus across the field question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!Z7U94tM1sMtYZhhlvDZ6lwfd94ioWz9rYEzmfx_koBo2Yd27Vv0vHrKYYJniWkf8v_EU$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!Z7U94tM1sMtYZhhlvDZ6lwfd94ioWz9rYEzmfx_koBo2Yd27Vv0vHrKYYJniWjtWmxze$
> [imgur[.]com] and include the link in your posting.
> *****
>
> Dear confocalers and other light microscopists,
>
> We have a question about tilt of a specimen or detectors leading to shift
> of focus across a field.
>
> From the first confocal microscope we used in 1991 through many other
> microscopes over the years, we have not had problems with planapochromat
> lenses with the image being in focus corner to corner across the field.
> This includes newer microscopes with wider fields of view and a spinning
> disk confocal with a Cairn twin camera setup.  (The cameras, however,
> required substantial alignment to attain this, but it has been locked in
> place for over a year.)  We are used to putting in standard stage inserts
> or even unusual ones, a variety of samples, and being able to focus at the
> substrate across the entire field.  This includes imaging in reflection
> mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent
> confocal.  This is routine.
>
> We have a new microscope, however, which does not have this focus
> uniformity.  I believe it is a dual camera alignment issue and we have been
> discussing this for almost a year as an installation issue, but recently
> the manufacturer insists that the only way to fix the problem we need to
> use screws on the stage to adjust the tilt.  I am concerned that this is
> going to be a major problem in a core facility with users who will play
> with anything.  It is going to add substantial burden on staff.  Also, it
> means that standard inserts will not work.  And it makes me wonder what
> this will do to the PSF if the coverglass isn't perpendicular to the light
> path.
>
> Have we been unusually lucky all these years to never have encountered
> this problem before?  Are we unreasonable to expect the microscope to be
> installed with all planes parallel for routine use?  Is this type of stage
> alignment standard acceptable procedure in other labs?
>
> Thank you.
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
>
> [hidden email]<mailto:[hidden email]>
> https://urldefense.com/v3/__http://nyulmc.org/micros__;!!CjcC7IQ!Z7U94tM1sMtYZhhlvDZ6lwfd94ioWz9rYEzmfx_koBo2Yd27Vv0vHrKYYJniWk7qagUc$
> [nyulmc[.]org]
> https://urldefense.com/v3/__http://microscopynotes.com/__;!!CjcC7IQ!Z7U94tM1sMtYZhhlvDZ6lwfd94ioWz9rYEzmfx_koBo2Yd27Vv0vHrKYYJniWvaaQUMl$
> [microscopynotes[.]com]
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
> ------------------------------------------------------------
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> intended recipient(s) and may contain information that is proprietary,
> confidential, and exempt from disclosure under applicable law. Any
> unauthorized review, use, disclosure, or distribution is prohibited. If you
> have received this email in error please notify the sender by return email
> and delete the original message. Please note, the recipient should check
> this email and any attachments for the presence of viruses. The
> organization accepts no liability for any damage caused by any virus
> transmitted by this email.
> =================================
>
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[Armstrong, Brian]

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Hi Michael, for our Zeiss confocal with Airyscan we have a leveling stage insert that came with a protocol for using it. If you want to spend the time of a few minutes you can get a small field of view very flat. Some stage inserts are fairly uniform across vendors so perhaps this could be adapted to your system.

Cheers,

Brian Armstrong PhD
Director, Light Microscopy Core
Research Professor
Department of Shared Resources
Beckman Research Institute, City of Hope

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael
Sent: Tuesday, June 15, 2021 6:21 PM
To: [hidden email]
Subject: uniform focus across the field question

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Dear confocalers and other light microscopists,

We have a question about tilt of a specimen or detectors leading to shift of focus across a field.

From the first confocal microscope we used in 1991 through many other microscopes over the years, we have not had problems with planapochromat lenses with the image being in focus corner to corner across the field.  This includes newer microscopes with wider fields of view and a spinning disk confocal with a Cairn twin camera setup.  (The cameras, however, required substantial alignment to attain this, but it has been locked in place for over a year.)  We are used to putting in standard stage inserts or even unusual ones, a variety of samples, and being able to focus at the substrate across the entire field.  This includes imaging in reflection mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent confocal.  This is routine.

We have a new microscope, however, which does not have this focus uniformity.  I believe it is a dual camera alignment issue and we have been discussing this for almost a year as an installation issue, but recently the manufacturer insists that the only way to fix the problem we need to use screws on the stage to adjust the tilt.  I am concerned that this is going to be a major problem in a core facility with users who will play with anything.  It is going to add substantial burden on staff.  Also, it means that standard inserts will not work.  And it makes me wonder what this will do to the PSF if the coverglass isn't perpendicular to the light path.

Have we been unusually lucky all these years to never have encountered this problem before?  Are we unreasonable to expect the microscope to be installed with all planes parallel for routine use?  Is this type of stage alignment standard acceptable procedure in other labs?

Thank you.



Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

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[Steffen Dietzel]

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Hi Michael,

does your microscope have eye pieces? If so, checking for a flat field
with the eye pieces should solve the issue whether the tilt comes from
the camera alignment or from the stage, no? Beads on a glass surface or
any other thin sample should be a good sample for this.

Also, how does the tilt behave with different objectives, say 40x and
100x? You should get a different outcome depending on whether the
problems arise from the sample/stage or behind the intermediate image.

Good hunting!

Steffen

Am 16.06.2021 um 03:21 schrieb Cammer, Michael:

> *****
> 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.
> *****
>
> Dear confocalers and other light microscopists,
>
> We have a question about tilt of a specimen or detectors leading to shift of focus across a field.
>
> >From the first confocal microscope we used in 1991 through many other microscopes over the years, we have not had problems with planapochromat lenses with the image being in focus corner to corner across the field.  This includes newer microscopes with wider fields of view and a spinning disk confocal with a Cairn twin camera setup.  (The cameras, however, required substantial alignment to attain this, but it has been locked in place for over a year.)  We are used to putting in standard stage inserts or even unusual ones, a variety of samples, and being able to focus at the substrate across the entire field.  This includes imaging in reflection mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent confocal.  This is routine.
>
> We have a new microscope, however, which does not have this focus uniformity.  I believe it is a dual camera alignment issue and we have been discussing this for almost a year as an installation issue, but recently the manufacturer insists that the only way to fix the problem we need to use screws on the stage to adjust the tilt.  I am concerned that this is going to be a major problem in a core facility with users who will play with anything.  It is going to add substantial burden on staff.  Also, it means that standard inserts will not work.  And it makes me wonder what this will do to the PSF if the coverglass isn't perpendicular to the light path.
>
> Have we been unusually lucky all these years to never have encountered this problem before?  Are we unreasonable to expect the microscope to be installed with all planes parallel for routine use?  Is this type of stage alignment standard acceptable procedure in other labs?
>
> Thank you.
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
>
> [hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Zdenek Svindrych-2]

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Or (seconding to Steffen), does the microscope stand have another pot where
you could connect a camera directly?
As already mentioned, if your sample does not go too much out of focus when
you move the stage a long distance (1 cm) then you can't fix your issue
with stage insert leveling screws. The issue then must be somewhere between
the objective lens and the camera (filters in the main turret are not
suspect, as these are in transmission and in infinity space; thanks Dan for
pointing out the nosepiece).
Alternatively, the whole XY stage is not bolted correctly to the microscope
frame.
zdenek

On Wed, Jun 16, 2021 at 12:51 PM Steffen Dietzel <[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 Michael,
>
> does your microscope have eye pieces? If so, checking for a flat field
> with the eye pieces should solve the issue whether the tilt comes from
> the camera alignment or from the stage, no? Beads on a glass surface or
> any other thin sample should be a good sample for this.
>
> Also, how does the tilt behave with different objectives, say 40x and
> 100x? You should get a different outcome depending on whether the
> problems arise from the sample/stage or behind the intermediate image.
>
> Good hunting!
>
> Steffen
>
> Am 16.06.2021 um 03:21 schrieb Cammer, Michael:
> > *****
> > 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.
> > *****
> >
> > Dear confocalers and other light microscopists,
> >
> > We have a question about tilt of a specimen or detectors leading to
> shift of focus across a field.
> >
> > >From the first confocal microscope we used in 1991 through many other
> microscopes over the years, we have not had problems with planapochromat
> lenses with the image being in focus corner to corner across the field.
> This includes newer microscopes with wider fields of view and a spinning
> disk confocal with a Cairn twin camera setup.  (The cameras, however,
> required substantial alignment to attain this, but it has been locked in
> place for over a year.)  We are used to putting in standard stage inserts
> or even unusual ones, a variety of samples, and being able to focus at the
> substrate across the entire field.  This includes imaging in reflection
> mode and TIRF in addition to Nomarski, epi-fluorescence, and fluorescent
> confocal.  This is routine.
> >
> > We have a new microscope, however, which does not have this focus
> uniformity.  I believe it is a dual camera alignment issue and we have been
> discussing this for almost a year as an installation issue, but recently
> the manufacturer insists that the only way to fix the problem we need to
> use screws on the stage to adjust the tilt.  I am concerned that this is
> going to be a major problem in a core facility with users who will play
> with anything.  It is going to add substantial burden on staff.  Also, it
> means that standard inserts will not work.  And it makes me wonder what
> this will do to the PSF if the coverglass isn't perpendicular to the light
> path.
> >
> > Have we been unusually lucky all these years to never have encountered
> this problem before?  Are we unreasonable to expect the microscope to be
> installed with all planes parallel for routine use?  Is this type of stage
> alignment standard acceptable procedure in other labs?
> >
> > Thank you.
> >
> >
> >
> > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> >
> > NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York,
> NY  10016
> >
> > [hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/
> >
> > Voice direct only, no text or messages:  1-914-309-3270 and
> 1-646-501-0567
> >
> >
> > ------------------------------------------------------------
> > This email message, including any attachments, is for the sole use of
> the intended recipient(s) and may contain information that is proprietary,
> confidential, and exempt from disclosure under applicable law. Any
> unauthorized review, use, disclosure, or distribution is prohibited. If you
> have received this email in error please notify the sender by return email
> and delete the original message. Please note, the recipient should check
> this email and any attachments for the presence of viruses. The
> organization accepts no liability for any damage caused by any virus
> transmitted by this email.
> > =================================
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth