Nikon Perfect Focus System problem

[Andrew Vaughan]

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Dear all,

I wonder if anyone out there can help me with a problem I'm having with the
Perfect Focus System on a Nikon Ti. Basically, the PFS won't work when I use
my 100X VC Plan Apochromat objective. My specimens are in various vessels,
but all of them have coverslip thickness glass and at least 3mm of
water/medium. When I focus on a specimen the PFS focus indicator light on
the front of the stand indicates that the objective lens is not in the focusable
range, even when the objective is focussed on the very surface of the
coverslip. To test this I drew crosses in fine black marker pen on the inner
coverslip surface of various chamber types. I then filled the chambers with
water and focussed on the cross using transmitted light. My results are as
follows:

1. The failure of the PFS seems to be limited to the 100X VC Plan Apochromat.
Lower power lenses have consistently worked when tested.
2. If I move the stage so that the entire field is filled with the dark marker
pen, the focus indicator lights up and PFS works. This is true whether or not
the transmitted light is on or switched off.
3. If I get the PFS to work in the manner above and then move the stage so
that I’m looking at an area free of marker pen the PFS stays on unless I turn
the transmitted light off or put an IR blocking filter in front of the transmitted
light.
4. If I blindly (with the transmitted light off) move the stage back into an area
where there is marker pen, the focus indicator light comes back on and the
PFS will work.
5. Additionally, if I get the PFS working using the method above and then
move the stage into a clear area – while still observing using transmitted light
– and then try to adjust the offset using the PFS Offset Controller, this causes
the PFS to fail again.

From this I can say certain things:

1. Only the 100X VC Plan Apochromat is affected
2. A dark field (or possibly one with high contrast edges) is required for the
PFS to work initially
3. The PFS can be sustained by IR light coming from the halogen lamp, but
this isn't enough to get it going initially
4. Adjusting the offset, which moves the dichroic mirror in the PFS nosepiece,
seems to take the system out of the focusable range

Note that I can’t rule out a fault with the objective, but I have compared this
objective with another identical objective in other tests recently and have no
reason to believe it is faulty.

Any ideas or suggestions would be most welcome.

Thanks,

Andrew.

[Mario Faretta]

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Dear Andrew,
we noticed the same behaviour also with other objectives and the
hopinion I have is that the IR contribution from the transmitted light
avoid the sensor (that is in the far red region) to read correctly the
reflection from the glass. The reason to observe it only at high
magnification is probably connected to the increase in excitation that
you have to apply to the transmitted light source the improve the
contrast.  we solved it by blocking IR light or by reducing transmission
adjusting exposure time on the camera (consequently not working at the
ocular). You can also alternatively disactivate PFS from the software,
acquire transmitted light image, block transmission and reactivate PFS.
Hope it helps
Mario
Il 2/28/2012 2:07 PM, Andrew Vaughan ha scritto:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> I wonder if anyone out there can help me with a problem I'm having with the
> Perfect Focus System on a Nikon Ti. Basically, the PFS won't work when I use
> my 100X VC Plan Apochromat objective. My specimens are in various vessels,
> but all of them have coverslip thickness glass and at least 3mm of
> water/medium. When I focus on a specimen the PFS focus indicator light on
> the front of the stand indicates that the objective lens is not in the focusable
> range, even when the objective is focussed on the very surface of the
> coverslip. To test this I drew crosses in fine black marker pen on the inner
> coverslip surface of various chamber types. I then filled the chambers with
> water and focussed on the cross using transmitted light. My results are as
> follows:
>
> 1. The failure of the PFS seems to be limited to the 100X VC Plan Apochromat.
> Lower power lenses have consistently worked when tested.
> 2. If I move the stage so that the entire field is filled with the dark marker
> pen, the focus indicator lights up and PFS works. This is true whether or not
> the transmitted light is on or switched off.
> 3. If I get the PFS to work in the manner above and then move the stage so
> that I’m looking at an area free of marker pen the PFS stays on unless I turn
> the transmitted light off or put an IR blocking filter in front of the transmitted
> light.
> 4. If I blindly (with the transmitted light off) move the stage back into an area
> where there is marker pen, the focus indicator light comes back on and the
> PFS will work.
> 5. Additionally, if I get the PFS working using the method above and then
> move the stage into a clear area – while still observing using transmitted light
> – and then try to adjust the offset using the PFS Offset Controller, this causes
> the PFS to fail again.
>
>  From this I can say certain things:
>
> 1. Only the 100X VC Plan Apochromat is affected
> 2. A dark field (or possibly one with high contrast edges) is required for the
> PFS to work initially
> 3. The PFS can be sustained by IR light coming from the halogen lamp, but
> this isn't enough to get it going initially
> 4. Adjusting the offset, which moves the dichroic mirror in the PFS nosepiece,
> seems to take the system out of the focusable range
>
> Note that I can’t rule out a fault with the objective, but I have compared this
> objective with another identical objective in other tests recently and have no
> reason to believe it is faulty.
>
> Any ideas or suggestions would be most welcome.
>
> Thanks,
>
> Andrew.

--
---PLEASE Note the change in telephone number---

--
Mario Faretta
Department of Experimental Oncology
European Institute of Oncology
c/o IFOM-IEO Campus for Oncogenomics
via Adamello 16 20139 Milan Italy
Phone: ++39-0294375027
email: [hidden email]
http://www.ifom-ieo-campus.it

[Kurt Thorn]

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You should really have an IR blocking filter in the transmitted light
path. I suspect the IR from the halogen lamp is fooling the PFS into
thinking it's seeing the coverslip reflection when really it's just
seeing the IR from the transmitted light. At least in our scopes, Nikon
always puts an IR blocking filter in the transmitted light path to
prevent these problems.

Kurt

On 2/28/2012 5:07 AM, Andrew Vaughan wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> I wonder if anyone out there can help me with a problem I'm having with the
> Perfect Focus System on a Nikon Ti. Basically, the PFS won't work when I use
> my 100X VC Plan Apochromat objective. My specimens are in various vessels,
> but all of them have coverslip thickness glass and at least 3mm of
> water/medium. When I focus on a specimen the PFS focus indicator light on
> the front of the stand indicates that the objective lens is not in the focusable
> range, even when the objective is focussed on the very surface of the
> coverslip. To test this I drew crosses in fine black marker pen on the inner
> coverslip surface of various chamber types. I then filled the chambers with
> water and focussed on the cross using transmitted light. My results are as
> follows:
>
> 1. The failure of the PFS seems to be limited to the 100X VC Plan Apochromat.
> Lower power lenses have consistently worked when tested.
> 2. If I move the stage so that the entire field is filled with the dark marker
> pen, the focus indicator lights up and PFS works. This is true whether or not
> the transmitted light is on or switched off.
> 3. If I get the PFS to work in the manner above and then move the stage so
> that I’m looking at an area free of marker pen the PFS stays on unless I turn
> the transmitted light off or put an IR blocking filter in front of the transmitted
> light.
> 4. If I blindly (with the transmitted light off) move the stage back into an area
> where there is marker pen, the focus indicator light comes back on and the
> PFS will work.
> 5. Additionally, if I get the PFS working using the method above and then
> move the stage into a clear area – while still observing using transmitted light
> – and then try to adjust the offset using the PFS Offset Controller, this causes
> the PFS to fail again.
>
>  From this I can say certain things:
>
> 1. Only the 100X VC Plan Apochromat is affected
> 2. A dark field (or possibly one with high contrast edges) is required for the
> PFS to work initially
> 3. The PFS can be sustained by IR light coming from the halogen lamp, but
> this isn't enough to get it going initially
> 4. Adjusting the offset, which moves the dichroic mirror in the PFS nosepiece,
> seems to take the system out of the focusable range
>
> Note that I can’t rule out a fault with the objective, but I have compared this
> objective with another identical objective in other tests recently and have no
> reason to believe it is faulty.
>
> Any ideas or suggestions would be most welcome.
>
> Thanks,
>
> Andrew.
>

[Andrew Vaughan]

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First of all I'd like to thank everyone who contacted me about this on and off the
forum. IR interference with the PFS was not the problem. I installed IR cut filters
in both the trans and epi illumination paths and I still had the same problem. I've
had two communications that seem to suggest that the particular objective lens
I'm using (100X VC Plan Apochromat 1.4 NA) can cause problems for the PFS,
although it is on Nikon's list of PFS-compatible objective lenses. I've managed to
work around the problem by installing a 100X Plan Fluor 1.3 NA objective
instead. I'm still in communication with Nikon and I'm going to try to get to the
bottom of this.

[Mario Faretta]

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Dear all,
we are trying to image fixed cell spheroids (>100um diameter) on a leica
sp5 inverted confocal microscope. The high resolution objectives we use
have short but sufficient working distance to analyse the spheres if
they attached to the  coverslip.  However standard coatings as
polilysine are not efficient in immobilizing them (even if they can
initially adhere we loose them extensively during staining). We tried  
also particular gels as Q gel but we were not able to find a
reproducible protocol to have them clearly attached to the substrate  
instead of being simply embedded in the gel.
I know that new high NA water-immersion long working-distance objectives
could provide the simplest solution but unfortunately their cost is
quite limiting.
thanks for all the answers
Mario

--
---PLEASE Note the change in telephone number---

--
Mario Faretta
Department of Experimental Oncology
European Institute of Oncology
c/o IFOM-IEO Campus for Oncogenomics
via Adamello 16 20139 Milan Italy
Phone: ++39-0294375027
email: [hidden email]
http://www.ifom-ieo-campus.it

[Tobias Baskin]

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Hi,
        Have you tried to activate your coverslip by putting it in a
plasma for 30 sec to 1 min? When we do this, we find that the
polylysine coating works much better than when the coverslips are
only acid cleaned. Your friendly neighborhood EM lab should be able
to provide a plasma for you to try.

        Hope it helps,
                Tobias

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear all,
>we are trying to image fixed cell spheroids (>100um diameter) on a
>leica sp5 inverted confocal microscope. The high resolution
>objectives we use have short but sufficient working distance to
>analyse the spheres if they attached to the  coverslip.  However
>standard coatings as polilysine are not efficient in immobilizing
>them (even if they can initially adhere we loose them extensively
>during staining). We tried  also particular gels as Q gel but we
>were not able to find a reproducible protocol to have them clearly
>attached to the substrate  instead of being simply embedded in the
>gel.
>I know that new high NA water-immersion long working-distance
>objectives could provide the simplest solution but unfortunately
>their cost is quite limiting.
>thanks for all the answers
>Mario
>
>--
>---PLEASE Note the change in telephone number---
>
>--
>Mario Faretta
>Department of Experimental Oncology
>European Institute of Oncology
>c/o IFOM-IEO Campus for Oncogenomics
>via Adamello 16 20139 Milan Italy
>Phone: ++39-0294375027
>email: [hidden email]
>http://www.ifom-ieo-campus.it

--
       _      ____          __   ____  
      /  \   /          / \    /   \ \       Tobias I. Baskin
     /   /  /          /   \   \      \        Professor
    /_ /   __      /__ \   \       \__    Biology Department
   /      /          /       \   \       \        611 N. Pleasant St.
  /      /          /         \   \       \      University of
Massachusetts
/      / ___   /           \   \__/  \ ____ Amherst, MA, 01003
www.bio.umass.edu/biology/baskin
Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

["José A. Feijó"]

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Hi Tobias. what kind of plasma, like with argon or something? when you
mention EM, you're thinking about the gold sputter coating device? and
after, you still go through HF etching or straight to poly-L ?
good to hear from you
Jose

Em 12-03-2012 13:32, Tobias Baskin escreveu:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
> Have you tried to activate your coverslip by putting it in a
> plasma for 30 sec to 1 min? When we do this, we find that the
> polylysine coating works much better than when the coverslips are
> only acid cleaned. Your friendly neighborhood EM lab should be able
> to provide a plasma for you to try.
>
> Hope it helps,
> Tobias
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>> we are trying to image fixed cell spheroids (>100um diameter) on a
>> leica sp5 inverted confocal microscope. The high resolution
>> objectives we use have short but sufficient working distance to
>> analyse the spheres if they attached to the  coverslip.  However
>> standard coatings as polilysine are not efficient in immobilizing
>> them (even if they can initially adhere we loose them extensively
>> during staining). We tried  also particular gels as Q gel but we
>> were not able to find a reproducible protocol to have them clearly
>> attached to the substrate  instead of being simply embedded in the
>> gel.
>> I know that new high NA water-immersion long working-distance
>> objectives could provide the simplest solution but unfortunately
>> their cost is quite limiting.
>> thanks for all the answers
>> Mario
>>
>> --
>> ---PLEASE Note the change in telephone number---
>>
>> --
>> Mario Faretta
>> Department of Experimental Oncology
>> European Institute of Oncology
>> c/o IFOM-IEO Campus for Oncogenomics
>> via Adamello 16 20139 Milan Italy
>> Phone: ++39-0294375027
>> email: [hidden email]
>> http://www.ifom-ieo-campus.it
>

--


**********************************************************
Jose' A. Feijo', Professor Catedrático
----------------------------------------------------------
Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
PT-1749-016 Lisboa, PORTUGAL

tel. [phone]/24, fax  [phone]

and/ e

Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL

tel. [phone]/19, fax [phone]
__________________________________________________________
e.mail: [hidden email]  or  [hidden email]

URL: http://www.igc.gulbenkian.pt/research/unit/38
or   http://dbv.fc.ul.pt/users/jafeijo

**********************************************************

[Tobias Baskin]

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Hi,
        Ah, it is true that a sputter coater used
for SEM makes a plasma. This is actually fancier
than what is needed (and certainly you don't want
to sputter metal on your coverslip). But plasmas
are also used for cleaning surfaces. Apparently
they create ozone or something that attacks
hydrocarbons that coat everything.  It should be
possible to find a 'plasma cleaner' or 'etcher'
that is not a sputter coater. (Maybe on some
models of sputter coaters you could make a plasma
w/o coating but I think maybe not). EM labs use
them activate grids and other fighing of
hydroarbon menace. Materials Science labs might
have one, or anyone dealing with micro (nano!)
fabrication. The devices I have used are simple,
they have a small chamber whre you put your
coverslips or slides, and they pull a vacuum
(maybe 0.1 % of air, not sure exactly but nothing
too serious), and then generate a plasma in the
remaining air (I guess by using RF coils but I
don't know the engineering details). There is
nice blue/purple glow. This makes glass extremely
hydrophillic. This is useful for lots of things,
including polylysine coating.

         Right after plasma coating coverslips,
we add a drop of polylysine to the coverslip,
spreading if needed, and let it air dry. Then put
a generous drop of water on, let that air dry,
and the coverslips are ready. We use them as soon
as possible after that. The next day they are
less sticky.
        As ever
                Tobias

>
>
>Hi Tobias. what kind of plasma, like with argon
>or something? when you mention EM, you're
>thinking about the gold sputter coating device?
>and after, you still go through HF etching or
>straight to poly-L ?
>good to hear from you
>Jose
>
>Em 12-03-2012 13:32, Tobias Baskin escreveu:
>>
>>
>>Hi,
>> Have you tried to activate your coverslip by putting it in a
>>plasma for 30 sec to 1 min? When we do this, we find that the
>>polylysine coating works much better than when the coverslips are
>>only acid cleaned. Your friendly neighborhood EM lab should be able
>>to provide a plasma for you to try.
>>
>> Hope it helps,
>> Tobias
>>
>>>
>>>*****
>>>
>>>Dear all,
>>>we are trying to image fixed cell spheroids (>100um diameter) on a
>>>leica sp5 inverted confocal microscope. The high resolution
>>>objectives we use have short but sufficient working distance to
>>>analyse the spheres if they attached to the  coverslip.  However
>>>standard coatings as polilysine are not efficient in immobilizing
>>>them (even if they can initially adhere we loose them extensively
>>>during staining). We tried  also particular gels as Q gel but we
>>>were not able to find a reproducible protocol to have them clearly
>>>attached to the substrate  instead of being simply embedded in the
>>>gel.
>>>I know that new high NA water-immersion long working-distance
>>>objectives could provide the simplest solution but unfortunately
>>>their cost is quite limiting.
>>>thanks for all the answers
>>>Mario
>>>
>>>--
>>>---PLEASE Note the change in telephone number---
>>>
>>>--
>>>Mario Faretta
>>>Department of Experimental Oncology
>>>European Institute of Oncology
>>>c/o IFOM-IEO Campus for Oncogenomics
>>>via Adamello 16 20139 Milan Italy
>>>Phone: ++39-0294375027
>>>email: [hidden email]
>>
>
>--
>
>
>**********************************************************
>Jose' A. Feijo', Professor Catedrático
>----------------------------------------------------------
>Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>PT-1749-016 Lisboa, PORTUGAL
>
>tel. [phone]/24, fax  [phone]
>
>and/ e
>
>Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>
>tel. [phone]/19, fax [phone]
>__________________________________________________________
>e.mail: [hidden email]  or  [hidden email]
>
>
>**********************************************************

--
       _      ____          __   ____
      /  \   /          / \    /   \ \       Tobias I. Baskin
     /   /  /          /   \   \      \        Professor
    /_ /   __      /__ \   \       \__    Biology Department
   /      /          /       \   \       \        611 N. Pleasant St.
  /      /          /         \   \       \
University of Massachusetts
/      / ___   /           \   \__/  \ ____ Amherst, MA, 01003
www.bio.umass.edu/biology/baskin
Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

[Boswell, Carl A - (cboswell)]

Hi Mario,

Celltak is an excellent biological adhesive and would be particularly appropriate for your fixed preps.  It is the glue that marine mussels use to attach to rocks in the intertidal (read that "high wave action") zone.  There can be some problems with live cells as the adhesion is so strong that normal physiology can be disturbed.

http://www.bdbiosciences.com/external_files/dl/doc/manuals/live/web_enabled/354240_pug.pdf 

C

Carl A. Boswell
520-742-6131


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mario Faretta
Sent: Monday, March 12, 2012 5:42 AM
To: [hidden email]
Subject: mammospheres attachment

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Dear all,
we are trying to image fixed cell spheroids (>100um diameter) on a leica
sp5 inverted confocal microscope. The high resolution objectives we use have short but sufficient working distance to analyse the spheres if they attached to the  coverslip.  However standard coatings as polilysine are not efficient in immobilizing them (even if they can initially adhere we loose them extensively during staining). We tried also particular gels as Q gel but we were not able to find a reproducible protocol to have them clearly attached to the substrate instead of being simply embedded in the gel.
I know that new high NA water-immersion long working-distance objectives could provide the simplest solution but unfortunately their cost is quite limiting.
thanks for all the answers
Mario

--
---PLEASE Note the change in telephone number---

--
Mario Faretta
Department of Experimental Oncology
European Institute of Oncology
c/o IFOM-IEO Campus for Oncogenomics
via Adamello 16 20139 Milan Italy
Phone: ++39-0294375027
email: [hidden email]
http://www.ifom-ieo-campus.it