Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

[mcammer]

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A few weeks ago we asked about finding a light source at 780ish nm for exciting Cy7.5.  As always, people on this listserv provided very helpful suggestions.  We were able to get a pE-4000 system with multiple LEDs but are finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics appear to be cutting wavelengths above 700 nm.

We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52 mW at the end of the liquid light guide.  We removed the IR filter for the autofocus system that is in the objectives nosepiece.  However, the light at 740 and 770 nm is blocked from arriving at the objective lenses.

Does anyone know whether there is a filter somewhere else in the light path we could easily remove to pass these wavelengths or is this a limitation of the light train?

(graphs of the power loss posted at http://microscopynotes.com/axioobserver/IR/ )
Thank you!!

=*===========================================================*=
Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
    Cell:  914-309-3270 (this is for calling, not texting)    Office: Skirball 2nd Floor main office, back right
      http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/



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=================================

[Craig Brideau]

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Are you very sure your dichroics and excitation filters are appropriate for
the long wavelengths you are using? Some visible coating filters have
unusual spikes in the NIR because they were never intended to work in that
range. This is not uncommon among longpass filters, which can act more like
unpredictable bandpass filters if you are using them outside their stated
wavelength ranges.

Craig


On Tue, Jul 11, 2017 at 1:47 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.
> *****
>
> A few weeks ago we asked about finding a light source at 780ish nm for
> exciting Cy7.5.  As always, people on this listserv provided very helpful
> suggestions.  We were able to get a pE-4000 system with multiple LEDs but
> are finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics
> appear to be cutting wavelengths above 700 nm.
>
> We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52
> mW at the end of the liquid light guide.  We removed the IR filter for the
> autofocus system that is in the objectives nosepiece.  However, the light
> at 740 and 770 nm is blocked from arriving at the objective lenses.
>
> Does anyone know whether there is a filter somewhere else in the light
> path we could easily remove to pass these wavelengths or is this a
> limitation of the light train?
>
> (graphs of the power loss posted at http://microscopynotes.com/
> axioobserver/IR/ )
> Thank you!!
>
> =*===========================================================*=
> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
>     Cell:  914-309-3270 (this is for calling, not texting)    Office:
> Skirball 2nd Floor main office, back right
>       http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
>
> ------------------------------------------------------------
> 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.
> =================================
>

[Sven Terclavers-3]

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The light train itself has a transmission beyond 1100nm. I assume the (optional) IR filter you’re talking about is the one that sits just below the filter cubes in a slider, right above the tube lens. This one should not even affect illumination as it’s a filter for emission light. Have you checked whether there’s a small compensation glass sitting on top of the filter turret? I don’t know its specific transmission spectrum, but it might be that one. Since you have DF you can leave this out completely. Furthermore, no attenuator filters that could influence transmission? And as mentioned by Craig, which filters are you using? When you remove the filter turret, do you see the light coming through the light train?
Best regards,

Sven

Sent from Mail for Windows 10

From: Cammer, Michael
Sent: Tuesday, July 11, 2017 10:01 PM
To: [hidden email]
Subject: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

*****
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*****

A few weeks ago we asked about finding a light source at 780ish nm for exciting Cy7.5.  As always, people on this listserv provided very helpful suggestions.  We were able to get a pE-4000 system with multiple LEDs but are finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics appear to be cutting wavelengths above 700 nm.

We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52 mW at the end of the liquid light guide.  We removed the IR filter for the autofocus system that is in the objectives nosepiece.  However, the light at 740 and 770 nm is blocked from arriving at the objective lenses.

Does anyone know whether there is a filter somewhere else in the light path we could easily remove to pass these wavelengths or is this a limitation of the light train?

(graphs of the power loss posted at http://microscopynotes.com/axioobserver/IR/ )
Thank you!!

=*===========================================================*=
Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
    Cell:  914-309-3270 (this is for calling, not texting)    Office: Skirball 2nd Floor main office, back right
      http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/



------------------------------------------------------------
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=================================

[Zdenek Svindrych-2]

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Hi Michael,
if you get the same readings before the filter cube (i.e. with the filter
turret removed), then there might be an IR blocking (heat blocking) filter
somewhere in the illumination part.

According to this drawing

http://www.zebrasc.com/Article.asp?pageclass=10201

there is quite a number of elements in the light path. It could also be the
AR coating of the lenses (e.g Thorlabs VIS coating reflects up to 5% per
surface at 800nm), but the dramatic loss of intensity looks more like an
intentional IR blocking. Zeiss reps will know more...
Good luck!

zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/
[hidden email]

---------- Původní e-mail ----------
Od: Cammer, Michael <[hidden email]>
Komu: [hidden email]
Datum: 11. 7. 2017 22:01:28
Předmět: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
"*****
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.
*****

A few weeks ago we asked about finding a light source at 780ish nm for
exciting Cy7.5. As always, people on this listserv provided very helpful
suggestions. We were able to get a pE-4000 system with multiple LEDs but are
finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics
appear to be cutting wavelengths above 700 nm.

We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52
mW at the end of the liquid light guide. We removed the IR filter for the
autofocus system that is in the objectives nosepiece. However, the light at
740 and 770 nm is blocked from arriving at the objective lenses.

Does anyone know whether there is a filter somewhere else in the light path
we could easily remove to pass these wavelengths or is this a limitation of
the light train?

(graphs of the power loss posted at http://microscopynotes.com/axioobserver/
IR/ )
Thank you!!

=*===========================================================*=
Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball 2nd
Floor main office, back right
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/



------------------------------------------------------------
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.
=================================
"

[Benjamin Smith]

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As others have pointed out, the strong 700 nm cutoff sounds very much like
a hot-mirror somewhere in the path, as that is a very common cut-off for
hot mirrors:
https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6108
https://www.edmundoptics.com/optics/optical-mirrors/specialty-mirrors/hot-mirrors/#resources
https://www.chroma.com/products/parts/hot-mirror-26mm-50mm

The hot mirror should be close to the light entry port on the microscope.
Some microscopes allow the mirror to be flipped out with a lever, while on
others it has to be physically removed.  A Zeiss rep should know exactly
where it is in the path.

Good luck,
   Ben Smith


On Tue, Jul 11, 2017 at 7:30 PM, <[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,
> if you get the same readings before the filter cube (i.e. with the filter
> turret removed), then there might be an IR blocking (heat blocking) filter
> somewhere in the illumination part.
>
> According to this drawing
>
> http://www.zebrasc.com/Article.asp?pageclass=10201
>
> there is quite a number of elements in the light path. It could also be the
> AR coating of the lenses (e.g Thorlabs VIS coating reflects up to 5% per
> surface at 800nm), but the dramatic loss of intensity looks more like an
> intentional IR blocking. Zeiss reps will know more...
> Good luck!
>
> zdenek
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> Department of Biology,University of Virginia
> 409 McCormick Rd, Charlottesville, VA-22904
> http://www.kcci.virginia.edu/
> [hidden email]
>
> ---------- Původní e-mail ----------
> Od: Cammer, Michael <[hidden email]>
> Komu: [hidden email]
> Datum: 11. 7. 2017 22:01:28
> Předmět: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
> "*****
> 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.
> *****
>
> A few weeks ago we asked about finding a light source at 780ish nm for
> exciting Cy7.5. As always, people on this listserv provided very helpful
> suggestions. We were able to get a pE-4000 system with multiple LEDs but
> are
> finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics
> appear to be cutting wavelengths above 700 nm.
>
> We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52
> mW at the end of the liquid light guide. We removed the IR filter for the
> autofocus system that is in the objectives nosepiece. However, the light at
> 740 and 770 nm is blocked from arriving at the objective lenses.
>
> Does anyone know whether there is a filter somewhere else in the light path
> we could easily remove to pass these wavelengths or is this a limitation of
> the light train?
>
> (graphs of the power loss posted at http://microscopynotes.com/
> axioobserver/
> IR/ )
> Thank you!!
>
> =*===========================================================*=
> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
> Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball 2nd
> Floor main office, back right
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
>
> ------------------------------------------------------------
> 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.
> =================================
> "
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Steffen Dietzel]

*****
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.
*****

and if everything else should fail, you could try to couple the LED
fiber through the condenser into the light path and do transmission
fluorescence, as in the old days before the Ploem pak was invented in
the 60ies, at least for testing purposes.

But be careful: one of the major advantages of Epifluorescence is lost,
and that is usage safety: if you look into the light source through the
eye piece without appropriate filters in the beam path you may very well
damage your eyes. With above 700 nm, possibly you might not even notice
it at first. I am not sure how much damage 'unvisible' red light might do.

Steffen


Am 12.07.2017 um 07:18 schrieb Benjamin E Smith:

> *****
> 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.
> *****
>
> As others have pointed out, the strong 700 nm cutoff sounds very much like
> a hot-mirror somewhere in the path, as that is a very common cut-off for
> hot mirrors:
> https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6108
> https://www.edmundoptics.com/optics/optical-mirrors/specialty-mirrors/hot-mirrors/#resources
> https://www.chroma.com/products/parts/hot-mirror-26mm-50mm
>
> The hot mirror should be close to the light entry port on the microscope.
> Some microscopes allow the mirror to be flipped out with a lever, while on
> others it has to be physically removed.  A Zeiss rep should know exactly
> where it is in the path.
>
> Good luck,
>     Ben Smith
>
>
> On Tue, Jul 11, 2017 at 7:30 PM, <[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,
>> if you get the same readings before the filter cube (i.e. with the filter
>> turret removed), then there might be an IR blocking (heat blocking) filter
>> somewhere in the illumination part.
>>
>> According to this drawing
>>
>> http://www.zebrasc.com/Article.asp?pageclass=10201
>>
>> there is quite a number of elements in the light path. It could also be the
>> AR coating of the lenses (e.g Thorlabs VIS coating reflects up to 5% per
>> surface at 800nm), but the dramatic loss of intensity looks more like an
>> intentional IR blocking. Zeiss reps will know more...
>> Good luck!
>>
>> zdenek
>> --
>> Zdenek Svindrych, Ph.D.
>> W.M. Keck Center for Cellular Imaging (PLSB 003)
>> Department of Biology,University of Virginia
>> 409 McCormick Rd, Charlottesville, VA-22904
>> http://www.kcci.virginia.edu/
>> [hidden email]
>>
>> ---------- Původní e-mail ----------
>> Od: Cammer, Michael <[hidden email]>
>> Komu: [hidden email]
>> Datum: 11. 7. 2017 22:01:28
>> Předmět: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>> "*****
>> 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.
>> *****
>>
>> A few weeks ago we asked about finding a light source at 780ish nm for
>> exciting Cy7.5. As always, people on this listserv provided very helpful
>> suggestions. We were able to get a pE-4000 system with multiple LEDs but
>> are
>> finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics
>> appear to be cutting wavelengths above 700 nm.
>>
>> We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52
>> mW at the end of the liquid light guide. We removed the IR filter for the
>> autofocus system that is in the objectives nosepiece. However, the light at
>> 740 and 770 nm is blocked from arriving at the objective lenses.
>>
>> Does anyone know whether there is a filter somewhere else in the light path
>> we could easily remove to pass these wavelengths or is this a limitation of
>> the light train?
>>
>> (graphs of the power loss posted at http://microscopynotes.com/
>> axioobserver/
>> IR/ )
>> Thank you!!
>>
>> =*===========================================================*=
>> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
>> Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball 2nd
>> Floor main office, back right
>> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>>
>>
>>
>> ------------------------------------------------------------
>> 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

[David Claypool]

*****
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*****

Check to be sure that there is not a IR cut off filter installed in your camera. Some of these IR cut filters are easily removeable.

David J. Claypool
Micro Video Instruments
Digital Imaging Product Manager
11 Robbie Road/PO Box 518
Avon, MA 02322
office: 800-875-2041 x5221
cell: 603-809-5342
[hidden email]
www.mvi-inc.com

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Wednesday, July 12, 2017 6:08 AM
To: [hidden email]
Subject: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

*****
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.
*****

and if everything else should fail, you could try to couple the LED fiber through the condenser into the light path and do transmission fluorescence, as in the old days before the Ploem pak was invented in the 60ies, at least for testing purposes.

But be careful: one of the major advantages of Epifluorescence is lost, and that is usage safety: if you look into the light source through the eye piece without appropriate filters in the beam path you may very well damage your eyes. With above 700 nm, possibly you might not even notice it at first. I am not sure how much damage 'unvisible' red light might do.

Steffen


Am 12.07.2017 um 07:18 schrieb Benjamin E Smith:

> *****
> 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.
> *****
>
> As others have pointed out, the strong 700 nm cutoff sounds very much
> like a hot-mirror somewhere in the path, as that is a very common
> cut-off for hot mirrors:
> https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6108
> https://www.edmundoptics.com/optics/optical-mirrors/specialty-mirrors/
> hot-mirrors/#resources
> https://www.chroma.com/products/parts/hot-mirror-26mm-50mm
>
> The hot mirror should be close to the light entry port on the microscope.
> Some microscopes allow the mirror to be flipped out with a lever,
> while on others it has to be physically removed.  A Zeiss rep should
> know exactly where it is in the path.
>
> Good luck,
>     Ben Smith
>
>
> On Tue, Jul 11, 2017 at 7:30 PM, <[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,
>> if you get the same readings before the filter cube (i.e. with the
>> filter turret removed), then there might be an IR blocking (heat
>> blocking) filter somewhere in the illumination part.
>>
>> According to this drawing
>>
>> http://www.zebrasc.com/Article.asp?pageclass=10201
>>
>> there is quite a number of elements in the light path. It could also
>> be the AR coating of the lenses (e.g Thorlabs VIS coating reflects up
>> to 5% per surface at 800nm), but the dramatic loss of intensity looks
>> more like an intentional IR blocking. Zeiss reps will know more...
>> Good luck!
>>
>> zdenek
>> --
>> Zdenek Svindrych, Ph.D.
>> W.M. Keck Center for Cellular Imaging (PLSB 003) Department of
>> Biology,University of Virginia
>> 409 McCormick Rd, Charlottesville, VA-22904
>> http://www.kcci.virginia.edu/ [hidden email]
>>
>> ---------- Původní e-mail ----------
>> Od: Cammer, Michael <[hidden email]>
>> Komu: [hidden email]
>> Datum: 11. 7. 2017 22:01:28
>> Předmět: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>> "*****
>> 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.
>> *****
>>
>> A few weeks ago we asked about finding a light source at 780ish nm
>> for exciting Cy7.5. As always, people on this listserv provided very
>> helpful suggestions. We were able to get a pE-4000 system with
>> multiple LEDs but are finding that the Zeiss AxioObserver.Z1
>> fluorescence illumination optics appear to be cutting wavelengths
>> above 700 nm.
>>
>> We adjusted the LED powers such that 635, 660, 740, and 770 nm all
>> have 52 mW at the end of the liquid light guide. We removed the IR
>> filter for the autofocus system that is in the objectives nosepiece.
>> However, the light at
>> 740 and 770 nm is blocked from arriving at the objective lenses.
>>
>> Does anyone know whether there is a filter somewhere else in the
>> light path we could easily remove to pass these wavelengths or is
>> this a limitation of the light train?
>>
>> (graphs of the power loss posted at http://microscopynotes.com/ 
>> axioobserver/ IR/ ) Thank you!!
>>
>> =*===========================================================*=
>> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical
>> Center
>> Cell: 914-309-3270 (this is for calling, not texting) Office:
>> Skirball 2nd Floor main office, back right
>> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>>
>>
>>
>> ------------------------------------------------------------
>> 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

[mcammer]

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*****

Thank you for the replies.  Thank you for the diagram which I reposted at http://microscopynotes.com/axioobserver/IR/diagram.png with the part of the light path we're having a problem with.  

The light guide is plugged in to the fluorescence illumination optics at the back of the AxioObserver.  Then there is a 50/50 mirror in the filter block position to project to the back aperture of the lens.  We put the mirror in because the electronics of the AxioObserver won't allow the shutter to open if the side port is open which we would need to put the power meter probe in.  But we tested the mirror independently and  it works ok out to 770 nm at least, so the problem must be in the illumination optics path.  But Sven points out there may be a filter between the filter block and the objective, so I will look for that, but since we have the IR autofocus device, I don't see how this would be possible.

I'll try another scope, but this is the one we really need to use because of the camera and stage for tiling, neither which can be moved to another scope body.  Also, we cannot do customization to this stand that would prevent routine use because it is in microscopy core.  Hence why we're looking for a filter that might be simple to pop out.

Thanks.

=*===========================================================*=
 Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
    Cell:  914-309-3270 (this is for calling, not texting)    Office: Skirball 2nd Floor main office, back right
      http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/



-----Original Message-----
From: Cammer, Michael
Sent: Tuesday, July 11, 2017 10:31 PM
To: Confocal Microscopy List
Subject: RE: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

At this point, there are no filters (that we know of) in the light path.  The light guide is plugged in to the fluorescence illumination optics at the back of the AxioObserver.  Then there is a 50/50 mirror in the filter block position to project to the back aperture of the lens.  We put the mirror in because the electronics of the AxioObserver won't allow the shutter to open if the side port is open which we would need to put the power meter probe in.  But the mirror is working ok out to 770 nm at least, so I thought issue must be in the illumination optics path.  But Sven points out there may be a filter between the filter block and the objective, so I will look for that, but since we have the IR autofocus device, I don;t see how this would be possible.
Thanks.

_________________________________________
Michael Cammer, Optical Microscopy Specialist http://ocs.med.nyu.edu/microscopy http://microscopynotes.com/
Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Sven Terclavers [[hidden email]]
Sent: Tuesday, July 11, 2017 10:25 PM
To: [hidden email]
Subject: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

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*****

The light train itself has a transmission beyond 1100nm. I assume the (optional) IR filter you're talking about is the one that sits just below the filter cubes in a slider, right above the tube lens. This one should not even affect illumination as it's a filter for emission light. Have you checked whether there's a small compensation glass sitting on top of the filter turret? I don't know its specific transmission spectrum, but it might be that one. Since you have DF you can leave this out completely. Furthermore, no attenuator filters that could influence transmission? And as mentioned by Craig, which filters are you using? When you remove the filter turret, do you see the light coming through the light train?
Best regards,

Sven

Sent from Mail for Windows 10

From: Cammer, Michael
Sent: Tuesday, July 11, 2017 10:01 PM
To: [hidden email]
Subject: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=fx3r4jy5w6K4sPEa0-PB9em1PNLvB-VQyUtttIyBu8o&e=  and include the link in your posting.
*****

A few weeks ago we asked about finding a light source at 780ish nm for exciting Cy7.5.  As always, people on this listserv provided very helpful suggestions.  We were able to get a pE-4000 system with multiple LEDs but are finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics appear to be cutting wavelengths above 700 nm.

We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52 mW at the end of the liquid light guide.  We removed the IR filter for the autofocus system that is in the objectives nosepiece.  However, the light at 740 and 770 nm is blocked from arriving at the objective lenses.

Does anyone know whether there is a filter somewhere else in the light path we could easily remove to pass these wavelengths or is this a limitation of the light train?

(graphs of the power loss posted at https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_axioobserver_IR_&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=5H18aiZASsoKyCJ0CWz_vELNL-F_nJdbJz3htY0tpT8&e=  ) Thank you!!

=*===========================================================*=
Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
    Cell:  914-309-3270 (this is for calling, not texting)    Office: Skirball 2nd Floor main office, back right
      http://ocs.med.nyu.edu/microscopy & https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=YfHG4GsP2w6AWO_eV_YxwEJd0_-WuRVVzQoRmEHD-FA&e=



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[Zdenek Svindrych-2]

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*****

Michael,
there is a plastic filter holder BELOW the filter block. That is not in the
excitation light path. Our Axio setup is different, as we have an LSM 780
NLO coupled to the back port. But for a widefield setup it makes perfect
sense to include IR blocking filter somewhere in the lamp or in the
illumination path, for the fluorescence filters are often misbehaving in the
IR...

You can grab a (metric) screwdriver and explore for yourself, or (as
suggested several times) ask Zeiss.

Good luck!

zdenek

---------- Původní e-mail ----------
Od: Cammer, Michael <[hidden email]>
Komu: [hidden email]
Datum: 12. 7. 2017 9:30:29
Předmět: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
"*****
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.
*****

Thank you for the replies. Thank you for the diagram which I reposted at
http://microscopynotes.com/axioobserver/IR/diagram.png with the part of the
light path we're having a problem with.

The light guide is plugged in to the fluorescence illumination optics at the
back of the AxioObserver. Then there is a 50/50 mirror in the filter block
position to project to the back aperture of the lens. We put the mirror in
because the electronics of the AxioObserver won't allow the shutter to open
if the side port is open which we would need to put the power meter probe
in. But we tested the mirror independently and it works ok out to 770 nm at
least, so the problem must be in the illumination optics path. But Sven
points out there may be a filter between the filter block and the objective,
so I will look for that, but since we have the IR autofocus device, I don't
see how this would be possible.

I'll try another scope, but this is the one we really need to use because of
the camera and stage for tiling, neither which can be moved to another scope
body. Also, we cannot do customization to this stand that would prevent
routine use because it is in microscopy core. Hence why we're looking for a
filter that might be simple to pop out.

Thanks.

=*===========================================================*=
Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball 2nd
Floor main office, back right
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/



-----Original Message-----
From: Cammer, Michael
Sent: Tuesday, July 11, 2017 10:31 PM
To: Confocal Microscopy List
Subject: RE: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

At this point, there are no filters (that we know of) in the light path. The
light guide is plugged in to the fluorescence illumination optics at the
back of the AxioObserver. Then there is a 50/50 mirror in the filter block
position to project to the back aperture of the lens. We put the mirror in
because the electronics of the AxioObserver won't allow the shutter to open
if the side port is open which we would need to put the power meter probe
in. But the mirror is working ok out to 770 nm at least, so I thought issue
must be in the illumination optics path. But Sven points out there may be a
filter between the filter block and the objective, so I will look for that,
but since we have the IR autofocus device, I don;t see how this would be
possible.
Thanks.

_________________________________________
Michael Cammer, Optical Microscopy Specialist http://ocs.med.nyu.edu/
microscopy http://microscopynotes.com/
Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf
of Sven Terclavers [[hidden email]]
Sent: Tuesday, July 11, 2017 10:25 PM
To: [hidden email]
Subject: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

*****
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78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=DASw7UwNEEnOcBauPfKePh4akaFo79LUYETn4V224
zQ&e=
Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05
LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17
ECXlq3M4bCw&s=fx3r4jy5w6K4sPEa0-PB9em1PNLvB-VQyUtttIyBu8o&e= and include the
link in your posting.
*****

The light train itself has a transmission beyond 1100nm. I assume the
(optional) IR filter you're talking about is the one that sits just below
the filter cubes in a slider, right above the tube lens. This one should not
even affect illumination as it's a filter for emission light. Have you
checked whether there's a small compensation glass sitting on top of the
filter turret? I don't know its specific transmission spectrum, but it might
be that one. Since you have DF you can leave this out completely.
Furthermore, no attenuator filters that could influence transmission? And as
mentioned by Craig, which filters are you using? When you remove the filter
turret, do you see the light coming through the light train?
Best regards,

Sven

Sent from Mail for Windows 10

From: Cammer, Michael
Sent: Tuesday, July 11, 2017 10:01 PM
To: [hidden email]
Subject: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?

*****
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78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=DASw7UwNEEnOcBauPfKePh4akaFo79LUYETn4V224
zQ&e=
Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05
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ECXlq3M4bCw&s=fx3r4jy5w6K4sPEa0-PB9em1PNLvB-VQyUtttIyBu8o&e= and include the
link in your posting.
*****

A few weeks ago we asked about finding a light source at 780ish nm for
exciting Cy7.5. As always, people on this listserv provided very helpful
suggestions. We were able to get a pE-4000 system with multiple LEDs but are
finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics
appear to be cutting wavelengths above 700 nm.

We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52
mW at the end of the liquid light guide. We removed the IR filter for the
autofocus system that is in the objectives nosepiece. However, the light at
740 and 770 nm is blocked from arriving at the objective lenses.

Does anyone know whether there is a filter somewhere else in the light path
we could easily remove to pass these wavelengths or is this a limitation of
the light train?

(graphs of the power loss posted at https://urldefense.proofpoint.com/v2/
url?u=http-3A__microscopynotes.com_axioobserver_IR_&d=DQIFaQ&c=j5oPpO0eBH1
iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_
CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=5H18aiZASsoKyCJ0CWz_
vELNL-F_nJdbJz3htY0tpT8&e= ) Thank you!!

=*===========================================================*=
Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball 2nd
Floor main office, back right
http://ocs.med.nyu.edu/microscopy & https://urldefense.proofpoint.com/v2/
url?u=http-3A__microscopynotes.com_&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5
jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP
78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=YfHG4GsP2w6AWO_eV_YxwEJd0_-WuRVVzQoRmEHD-
FA&e=



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and delete the original message. Please note, the recipient should check
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email.
=================================

------------------------------------------------------------
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
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"

[Rusty Nicovich]

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*****

From my schematic, the Axio Observer has a heat filter right behind the
aperture diaphragm in the rear port.  This would be internal to the body of
the scope and unlikely to be accessible or removable.

Assuming it's this part number as listed on the Zeiss website:

https://www.micro-shop.zeiss.com/index.php?p=at&f=a&i=10051_400000001200100000&o&h=25&n=0&sd=467830-0000-000&l=en

and it's really typical KG 1 glass, this page has the transmission/blocking
curves:

https://www.ahf.de/art-Waermeschutzfilter_KG_1__D_25_mm;F01-125.html

The 50% cut-off for transmission is 740 nm, which may be bad news if you
are illuminating with 740 nm or 770 nm.

These KG 1 optics:

https://www.knightoptical.com/stock/optical-components/uvvisnir-optics/filters/short-pass-filters/kg1-heat-glass-filters/

have an even worse 50% cut-off at 716 nm.  Light Googling can't seem to
find more info on the Zeiss product transmission but it doesn't look like
good news if you're trying to illuminate with > 720 nm.  In any case, a
call to Zeiss is going to be the way to go here.

Thanks,
Rusty



On Wed, Jul 12, 2017 at 7:04 AM, <[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.
> *****
>
> Michael,
> there is a plastic filter holder BELOW the filter block. That is not in the
> excitation light path. Our Axio setup is different, as we have an LSM 780
> NLO coupled to the back port. But for a widefield setup it makes perfect
> sense to include IR blocking filter somewhere in the lamp or in the
> illumination path, for the fluorescence filters are often misbehaving in
> the
> IR...
>
> You can grab a (metric) screwdriver and explore for yourself, or (as
> suggested several times) ask Zeiss.
>
> Good luck!
>
> zdenek
>
> ---------- Původní e-mail ----------
> Od: Cammer, Michael <[hidden email]>
> Komu: [hidden email]
> Datum: 12. 7. 2017 9:30:29
> Předmět: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
> "*****
> 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.
> *****
>
> Thank you for the replies. Thank you for the diagram which I reposted at
> http://microscopynotes.com/axioobserver/IR/diagram.png with the part of
> the
> light path we're having a problem with.
>
> The light guide is plugged in to the fluorescence illumination optics at
> the
> back of the AxioObserver. Then there is a 50/50 mirror in the filter block
> position to project to the back aperture of the lens. We put the mirror in
> because the electronics of the AxioObserver won't allow the shutter to open
> if the side port is open which we would need to put the power meter probe
> in. But we tested the mirror independently and it works ok out to 770 nm at
> least, so the problem must be in the illumination optics path. But Sven
> points out there may be a filter between the filter block and the
> objective,
> so I will look for that, but since we have the IR autofocus device, I don't
> see how this would be possible.
>
> I'll try another scope, but this is the one we really need to use because
> of
> the camera and stage for tiling, neither which can be moved to another
> scope
> body. Also, we cannot do customization to this stand that would prevent
> routine use because it is in microscopy core. Hence why we're looking for a
> filter that might be simple to pop out.
>
> Thanks.
>
> =*===========================================================*=
> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
> Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball 2nd
> Floor main office, back right
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
>
> -----Original Message-----
> From: Cammer, Michael
> Sent: Tuesday, July 11, 2017 10:31 PM
> To: Confocal Microscopy List
> Subject: RE: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>
> At this point, there are no filters (that we know of) in the light path.
> The
> light guide is plugged in to the fluorescence illumination optics at the
> back of the AxioObserver. Then there is a 50/50 mirror in the filter block
> position to project to the back aperture of the lens. We put the mirror in
> because the electronics of the AxioObserver won't allow the shutter to open
> if the side port is open which we would need to put the power meter probe
> in. But the mirror is working ok out to 770 nm at least, so I thought issue
> must be in the illumination optics path. But Sven points out there may be a
> filter between the filter block and the objective, so I will look for that,
> but since we have the IR autofocus device, I don;t see how this would be
> possible.
> Thanks.
>
> _________________________________________
> Michael Cammer, Optical Microscopy Specialist http://ocs.med.nyu.edu/
> microscopy http://microscopynotes.com/
> Cell: [phone]
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf
> of Sven Terclavers [[hidden email]]
> Sent: Tuesday, July 11, 2017 10:25 PM
> To: [hidden email]
> Subject: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.
> umn.edu_cgi-2Dbin_
> wa-3FA0-3Dconfocalmicroscopy&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5
> jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=
> hwRGJZNcWAhP
> 78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=DASw7UwNEEnOcBauPfKePh4akaFo79
> LUYETn4V224
> zQ&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
> imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05
> LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17
> ECXlq3M4bCw&s=fx3r4jy5w6K4sPEa0-PB9em1PNLvB-VQyUtttIyBu8o&e= and include
> the
> link in your posting.
> *****
>
> The light train itself has a transmission beyond 1100nm. I assume the
> (optional) IR filter you're talking about is the one that sits just below
> the filter cubes in a slider, right above the tube lens. This one should
> not
> even affect illumination as it's a filter for emission light. Have you
> checked whether there's a small compensation glass sitting on top of the
> filter turret? I don't know its specific transmission spectrum, but it
> might
> be that one. Since you have DF you can leave this out completely.
> Furthermore, no attenuator filters that could influence transmission? And
> as
> mentioned by Craig, which filters are you using? When you remove the filter
> turret, do you see the light coming through the light train?
> Best regards,
>
> Sven
>
> Sent from Mail for Windows 10
>
> From: Cammer, Michael
> Sent: Tuesday, July 11, 2017 10:01 PM
> To: [hidden email]
> Subject: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.
> umn.edu_cgi-2Dbin_
> wa-3FA0-3Dconfocalmicroscopy&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5
> jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=
> hwRGJZNcWAhP
> 78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=DASw7UwNEEnOcBauPfKePh4akaFo79
> LUYETn4V224
> zQ&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
> imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05
> LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17
> ECXlq3M4bCw&s=fx3r4jy5w6K4sPEa0-PB9em1PNLvB-VQyUtttIyBu8o&e= and include
> the
> link in your posting.
> *****
>
> A few weeks ago we asked about finding a light source at 780ish nm for
> exciting Cy7.5. As always, people on this listserv provided very helpful
> suggestions. We were able to get a pE-4000 system with multiple LEDs but
> are
> finding that the Zeiss AxioObserver.Z1 fluorescence illumination optics
> appear to be cutting wavelengths above 700 nm.
>
> We adjusted the LED powers such that 635, 660, 740, and 770 nm all have 52
> mW at the end of the liquid light guide. We removed the IR filter for the
> autofocus system that is in the objectives nosepiece. However, the light at
> 740 and 770 nm is blocked from arriving at the objective lenses.
>
> Does anyone know whether there is a filter somewhere else in the light path
> we could easily remove to pass these wavelengths or is this a limitation of
> the light train?
>
> (graphs of the power loss posted at https://urldefense.proofpoint.com/v2/
> url?u=http-3A__microscopynotes.com_axioobserver_IR_&d=DQIFaQ&c=j5oPpO0eBH1
> iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_
> CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=
> 5H18aiZASsoKyCJ0CWz_
> vELNL-F_nJdbJz3htY0tpT8&e= ) Thank you!!
>
> =*===========================================================*=
> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
> Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball 2nd
> Floor main office, back right
> http://ocs.med.nyu.edu/microscopy & https://urldefense.proofpoint.com/v2/
> url?u=http-3A__microscopynotes.com_&d=DQIFaQ&c=
> j5oPpO0eBH1iio48DtsedbOBGmuw5
> jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=
> hwRGJZNcWAhP
> 78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=YfHG4GsP2w6AWO_eV_YxwEJd0_
> -WuRVVzQoRmEHD-
> FA&e=
>
>
>
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[mcammer]

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*****

I am waiting to hear from Zeiss to find out whether we can easily remove the heat filter(s) in the new Axioobserver.  

In the meantime, I found a Zeiss AxioPlan, removed the KG? filter from the back, and the 740 and 770 nm light goes through it fine.  

Thank you all for the feedback.

=*===========================================================*=
 Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
    Cell:  914-309-3270 (this is for calling, not texting)    Office: Skirball 2nd Floor main office, back right
      http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rusty Nicovich
Sent: Wednesday, July 12, 2017 5:51 PM
To: [hidden email]
Subject: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?


From my schematic, the Axio Observer has a heat filter right behind the aperture diaphragm in the rear port.  This would be internal to the body of the scope and unlikely to be accessible or removable.

Assuming it's this part number as listed on the Zeiss website:

https://urldefense.proofpoint.com/v2/url?u=https-3A__www.micro-2Dshop.zeiss.com_index.php-3Fp-3Dat-26f-3Da-26i-3D10051-5F400000001200100000-26o-26h-3D25-26n-3D0-26sd-3D467830-2D0000-2D000-26l-3Den&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=v-XN3q2ZaJvuNm5ZaAEqtVQ_q4XN3I3FdCy07-lt2HQ&e= 

and it's really typical KG 1 glass, this page has the transmission/blocking
curves:

https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ahf.de_art-2DWaermeschutzfilter-5FKG-5F1-5F-5FD-5F25-5Fmm-3BF01-2D125.html&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=-qcTfxL57QX1KanZkMOFlMP6K4tsQrLDoibE5l55Fb4&e= 

The 50% cut-off for transmission is 740 nm, which may be bad news if you are illuminating with 740 nm or 770 nm.

These KG 1 optics:

https://urldefense.proofpoint.com/v2/url?u=https-3A__www.knightoptical.com_stock_optical-2Dcomponents_uvvisnir-2Doptics_filters_short-2Dpass-2Dfilters_kg1-2Dheat-2Dglass-2Dfilters_&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=8OjWDiwz0JAjfQYGPKj0iraF1qY_P0R96tCz8XuBUls&e= 

have an even worse 50% cut-off at 716 nm.  Light Googling can't seem to find more info on the Zeiss product transmission but it doesn't look like good news if you're trying to illuminate with > 720 nm.  In any case, a call to Zeiss is going to be the way to go here.

Thanks,
Rusty



On Wed, Jul 12, 2017 at 7:04 AM, <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOB
> Gmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2d
> d3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=0WY3yY_41mhJSk1zCEUYU-9eZU
> 7kPT-jGqG4gtnOkg8&e= Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=2xj5uVgfe650G4-dPx6A1GKU1-NFHn-iU2WnI98yjDA&e=  and include the link in your posting.
> *****
>
> Michael,
> there is a plastic filter holder BELOW the filter block. That is not
> in the excitation light path. Our Axio setup is different, as we have
> an LSM 780 NLO coupled to the back port. But for a widefield setup it
> makes perfect sense to include IR blocking filter somewhere in the
> lamp or in the illumination path, for the fluorescence filters are
> often misbehaving in the IR...
>
> You can grab a (metric) screwdriver and explore for yourself, or (as
> suggested several times) ask Zeiss.
>
> Good luck!
>
> zdenek
>
> ---------- Původní e-mail ----------
> Od: Cammer, Michael <[hidden email]>
> Komu: [hidden email]
> Datum: 12. 7. 2017 9:30:29
> Předmět: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOB
> Gmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2d
> d3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=0WY3yY_41mhJSk1zCEUYU-9eZU
> 7kPT-jGqG4gtnOkg8&e= Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=2xj5uVgfe650G4-dPx6A1GKU1-NFHn-iU2WnI98yjDA&e=  and include the link in your posting.
> *****
>
> Thank you for the replies. Thank you for the diagram which I reposted
> at
> https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.co
> m_axioobserver_IR_diagram.png&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng6VNFWuWi0&s=dqDcUpwkpA9gx4zh4bYfwfALTKNv8TvTKy_XRIapAWk&e=  with the part of the light path we're having a problem with.
>
> The light guide is plugged in to the fluorescence illumination optics
> at the back of the AxioObserver. Then there is a 50/50 mirror in the
> filter block position to project to the back aperture of the lens. We
> put the mirror in because the electronics of the AxioObserver won't
> allow the shutter to open if the side port is open which we would need
> to put the power meter probe in. But we tested the mirror
> independently and it works ok out to 770 nm at least, so the problem
> must be in the illumination optics path. But Sven points out there may
> be a filter between the filter block and the objective, so I will look
> for that, but since we have the IR autofocus device, I don't see how
> this would be possible.
>
> I'll try another scope, but this is the one we really need to use
> because of the camera and stage for tiling, neither which can be moved
> to another scope body. Also, we cannot do customization to this stand
> that would prevent routine use because it is in microscopy core. Hence
> why we're looking for a filter that might be simple to pop out.
>
> Thanks.
>
> =*===========================================================*=
> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
> Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball
> 2nd Floor main office, back right http://ocs.med.nyu.edu/microscopy &
> https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.co
> m_&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNs
> tAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng
> 6VNFWuWi0&s=QZ4D6s7slhBbAFuZqIi_9Wc5cwUtkaI-MnjamzzrURY&e=
>
>
>
> -----Original Message-----
> From: Cammer, Michael
> Sent: Tuesday, July 11, 2017 10:31 PM
> To: Confocal Microscopy List
> Subject: RE: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>
> At this point, there are no filters (that we know of) in the light path.
> The
> light guide is plugged in to the fluorescence illumination optics at
> the back of the AxioObserver. Then there is a 50/50 mirror in the
> filter block position to project to the back aperture of the lens. We
> put the mirror in because the electronics of the AxioObserver won't
> allow the shutter to open if the side port is open which we would need
> to put the power meter probe in. But the mirror is working ok out to
> 770 nm at least, so I thought issue must be in the illumination optics
> path. But Sven points out there may be a filter between the filter
> block and the objective, so I will look for that, but since we have
> the IR autofocus device, I don;t see how this would be possible.
> Thanks.
>
> _________________________________________
> Michael Cammer, Optical Microscopy Specialist http://ocs.med.nyu.edu/ 
> microscopy
> https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.co
> m_&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNs
> tAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=2dd3bRi4R-tIWosCp6IoMx8t8cNYhoiAng
> 6VNFWuWi0&s=QZ4D6s7slhBbAFuZqIi_9Wc5cwUtkaI-MnjamzzrURY&e=
> Cell: [phone]
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf of Sven Terclavers [[hidden email]]
> Sent: Tuesday, July 11, 2017 10:25 PM
> To: [hidden email]
> Subject: Re: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.
> umn.edu_cgi-2Dbin_
> wa-3FA0-3Dconfocalmicroscopy&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5
> jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=
> hwRGJZNcWAhP
> 78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=DASw7UwNEEnOcBauPfKePh4akaFo79
> LUYETn4V224
> zQ&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
> imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_
> 05
> LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2
> m17 ECXlq3M4bCw&s=fx3r4jy5w6K4sPEa0-PB9em1PNLvB-VQyUtttIyBu8o&e= and
> include the link in your posting.
> *****
>
> The light train itself has a transmission beyond 1100nm. I assume the
> (optional) IR filter you're talking about is the one that sits just
> below the filter cubes in a slider, right above the tube lens. This
> one should not even affect illumination as it's a filter for emission
> light. Have you checked whether there's a small compensation glass
> sitting on top of the filter turret? I don't know its specific
> transmission spectrum, but it might be that one. Since you have DF you
> can leave this out completely.
> Furthermore, no attenuator filters that could influence transmission?
> And as mentioned by Craig, which filters are you using? When you
> remove the filter turret, do you see the light coming through the
> light train?
> Best regards,
>
> Sven
>
> Sent from Mail for Windows 10
>
> From: Cammer, Michael
> Sent: Tuesday, July 11, 2017 10:01 PM
> To: [hidden email]
> Subject: Trying to image Cy7.5 -- AxioObserver.Z1 blocking IR?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.
> umn.edu_cgi-2Dbin_
> wa-3FA0-3Dconfocalmicroscopy&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5
> jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=
> hwRGJZNcWAhP
> 78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=DASw7UwNEEnOcBauPfKePh4akaFo79
> LUYETn4V224
> zQ&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.
> imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_
> 05
> LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2
> m17 ECXlq3M4bCw&s=fx3r4jy5w6K4sPEa0-PB9em1PNLvB-VQyUtttIyBu8o&e= and
> include the link in your posting.
> *****
>
> A few weeks ago we asked about finding a light source at 780ish nm for
> exciting Cy7.5. As always, people on this listserv provided very
> helpful suggestions. We were able to get a pE-4000 system with
> multiple LEDs but are finding that the Zeiss AxioObserver.Z1
> fluorescence illumination optics appear to be cutting wavelengths
> above 700 nm.
>
> We adjusted the LED powers such that 635, 660, 740, and 770 nm all
> have 52 mW at the end of the liquid light guide. We removed the IR
> filter for the autofocus system that is in the objectives nosepiece.
> However, the light at
> 740 and 770 nm is blocked from arriving at the objective lenses.
>
> Does anyone know whether there is a filter somewhere else in the light
> path we could easily remove to pass these wavelengths or is this a
> limitation of the light train?
>
> (graphs of the power loss posted at
> https://urldefense.proofpoint.com/v2/
> url?u=http-3A__microscopynotes.com_axioobserver_IR_&d=DQIFaQ&c=j5oPpO0
> eBH1
> iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDL
> x_ CqKkuo&m=hwRGJZNcWAhP78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=
> 5H18aiZASsoKyCJ0CWz_
> vELNL-F_nJdbJz3htY0tpT8&e= ) Thank you!!
>
> =*===========================================================*=
> Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
> Cell: 914-309-3270 (this is for calling, not texting) Office: Skirball
> 2nd Floor main office, back right http://ocs.med.nyu.edu/microscopy &
> https://urldefense.proofpoint.com/v2/
> url?u=http-3A__microscopynotes.com_&d=DQIFaQ&c=
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> hwRGJZNcWAhP
> 78SlBGjpWK7aUX3i2m17ECXlq3M4bCw&s=YfHG4GsP2w6AWO_eV_YxwEJd0_
> -WuRVVzQoRmEHD-
> FA&e=
>
>
>
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> 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
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>
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> law. Any unauthorized review, use, disclosure, or distribution is
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[Arvydas Matiukas]

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Dear microscopists,
 
We are building STED microscope, and now are at the step of
aligning 488nm excitation and 580nm depletion beams in the focal plane
by imaging gold 100nm beads (from Sigma). Surprisingly it was difficult
to see the reflected signal off the beads either visually, using CCD camera,
or APD detector (while 83nm green fluorescent beads produced a nice image).
So far  I was able to see the beads in a 1uL drop of stock solution squeezed
between coverslip and slide (visually or with CCD camera at slightly defocussed 580 laser
beam) . However they were violently moving due to Brownian
motion. Diluting and immobilizing beads in agarose made them "dissappear".
 
Please share your experience of imaging gold nanobeads (~100nm in diameter).
Specifically I have three questions:
1. Is there a preference for a specific manufacturer ? Do the beads degrade with time
(my are not fresh but the  bead stock still has pale red color).
2. Please share a bead slide preparation protocol that is working.
3. Any advise on illumination and detection settings? How bright the bead reflection
signal is expected to be?
 
 
Thanks,
Arvydas
 
 
 


 
 
 
*******************
Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept

Room 4607 IHP
505 Irving Ave
Syracuse, NY 13210

[Satoru Uzawa]

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Hi,

   When I imaged 15 nm gold beads on Leica SP2, I had to use hardening mounting media (I tried both ProLong Gold and Vectashield and ended up using ProLong Gold) to “fix” the gold beads.  Glycerol based mounting media are useless here.  Also, agarose will not make individual beads immobile.  Many beads got stuck to either the coverslip or to the slide glass, but I could find some that were in the middle.  Gold beads stuck on  the coverslip are useless, since the reflection form the glass surface will dominate the signal.  

   I used 12 uL of mounting media with series of dilution of the gold beads to mount with 18x18 mm coverslips. Vortex hard before you mount them.  If you can sonicate them, that’s better.  I hanged the slide upside down while they were curing to increase the distance from coverslip to slide (to be honest, I don’t know how much it helped, though).  As far as I can tell, the slide lasted quite long time, more than a month.  

  I would start with low dose of laser with PMT, but not with HyD or APD.  To find beads, you can start at the surface of the coverslip and move slightly into the mounting media where you will find some beads on a slide with high concentration of beads.  The faintest stable signal there would be from a single bead.  Once you know the intensity from the single beads, you can search one further inside the mounting media on a slide with lower concentration of beads.  In your case, it should be easier to find beads as they are substantially larger than what I used. I noticed that you may not need to identify any single gold bead, so you could just go with high concentration of beads.  I don’t think the distributor/manufacturer of the gold beads makes any difference for imaging.

   Good luck!

Best,

Satoru
______________

Satoru Uzawa
Meyer lab, HHMI/U.C. Berkeley
Dept. of MCB
131 Koshland
Berkeley, CA  94720-3204

> On Jul 17, 2017, at 10:55 AM, Arvydas Matiukas <[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 microscopists,
>
> We are building STED microscope, and now are at the step of
> aligning 488nm excitation and 580nm depletion beams in the focal plane
> by imaging gold 100nm beads (from Sigma). Surprisingly it was difficult
> to see the reflected signal off the beads either visually, using CCD camera,
> or APD detector (while 83nm green fluorescent beads produced a nice image).
> So far  I was able to see the beads in a 1uL drop of stock solution squeezed
> between coverslip and slide (visually or with CCD camera at slightly defocussed 580 laser
> beam) . However they were violently moving due to Brownian
> motion. Diluting and immobilizing beads in agarose made them "dissappear".
>
> Please share your experience of imaging gold nanobeads (~100nm in diameter).
> Specifically I have three questions:
> 1. Is there a preference for a specific manufacturer ? Do the beads degrade with time
> (my are not fresh but the  bead stock still has pale red color).
> 2. Please share a bead slide preparation protocol that is working.
> 3. Any advise on illumination and detection settings? How bright the bead reflection
> signal is expected to be?
>
>
> Thanks,
> Arvydas
>
>
>
>
>
>
>
>
> *******************
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> SUNY Upstate Medical University
> Neuroscience & Physiology Dept
>
> Room 4607 IHP
> 505 Irving Ave
> Syracuse, NY 13210