flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

[Sylvie Le Guyader]

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*****

Dear everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Csúcs Gábor-3]

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*****

Dear Sylvie,

I will need to check on the plate issue, but for the oils: we have been always using Zeiss oil on all of our microscopes (Zeiss, Leica, Olympus, Nikon). So far (over a couple of years) with no observable effect.
Concerning the plates: perhaps you could share your list of plates that you have tried already (without success).

Greetings    Gabor



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Dienstag, 16. Februar 2016 11:47
To: [hidden email]
Subject: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
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 everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Eric Griffis]

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*****

Hi Sylvie,

We've had good success with plates from Matrical (which may now be sold as Brooks?). The wells are square, so you have more surface area, and I've found that they have less toxicity compared to some plates from other vendors, which were more variable. They will also usually send you a sample plate to test in your application.

Hope this is helpful.

Best regards,

Eric



Centre for Gene Regulation and Expression
College of Life Sciences
University of Dundee
MSI/WTB/JBC Complex
Dow Street
Dundee DD1 5EH
United KIngdom

[phone]
[hidden email]<mailto:[hidden email]>
http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html



On 16 Feb 2016, at 10:46, Sylvie Le Guyader <[hidden email]<mailto:[hidden email]>>
 wrote:

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Dear everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>


The University of Dundee is a registered Scottish Charity, No: SC015096

[Sebastian Munck]

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*****

Dear Sylvie,

we have noticed enhanced (chromatic) aberrations, when applying the Zeiss oil on a Nikon confocal when using a high NA lens. Likely reflecting the difference in refractive index between the oils. You can test this with some fluorescent beads (easier visible with bigger ones).
Best Seb

________________________________________
Von: Confocal Microscopy List [[hidden email]]&quot; im Auftrag von &quot;Csúcs  Gábor [[hidden email]]
Gesendet: Dienstag, 16. Februar 2016 13:13
An: [hidden email]
Betreff: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear Sylvie,

I will need to check on the plate issue, but for the oils: we have been always using Zeiss oil on all of our microscopes (Zeiss, Leica, Olympus, Nikon). So far (over a couple of years) with no observable effect.
Concerning the plates: perhaps you could share your list of plates that you have tried already (without success).

Greetings    Gabor



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Dienstag, 16. Februar 2016 11:47
To: [hidden email]
Subject: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
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 everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Gary Laevsky]

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*****

Hi Sylvie,

Can't help with the plate, but, for use with Ibidi dishes and our Nikon 20X
MM objective we use Leica oil.  That objective doesn't live on the scope
(but it gets used weekly), and ONLY gets Leica oil.

Good luck.

Gary

On Tue, Feb 16, 2016 at 7:13 AM, Csúcs Gábor <[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 Sylvie,
>
> I will need to check on the plate issue, but for the oils: we have been
> always using Zeiss oil on all of our microscopes (Zeiss, Leica, Olympus,
> Nikon). So far (over a couple of years) with no observable effect.
> Concerning the plates: perhaps you could share your list of plates that
> you have tried already (without success).
>
> Greetings    Gabor
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Sylvie Le Guyader
> Sent: Dienstag, 16. Februar 2016 11:47
> To: [hidden email]
> Subject: flat glass bottom multiwell plates and Zeiss oil on Nikon
> objectives
>
> *****
> 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 everyone on the list
>
> I have 2 questions:
>
> 1-      It proves surprisingly difficult to find multiwell plates that
> work well for our microscopy purpose which is high res microscopy on
> multiple wells.
> Here are our requirements:
> - 96 well plates
> - borosilicate bottom
> - thickness #1.5 (170 or 175 um)
> - no skirt as it makes it impossible to image the outer wells with wide
> objectives
> - flat (ie no slant of a full field of view with a 10x objective)
>
> None of the plates we tried are actually flat. The glass is good but the
> plastic part is so slanted or distorted that we see the slope even in a
> single field of view so using a hardware autofocus doesn't help. Many
> plates have a glass bottom of thickness 145 um which is the wrong
> specification for the objectives. Some plates leak. Some kill the cells in
> about 30% of the wells!
>
> Could people on the list and vendors recommend glass bottom plates that
> fit with the requirements above?
>
>
> 2-      My second question is related. We have many Nikon microscopes. The
> oil Nikon sells for their objectives melts the plastic used in many
> 'optical' plastic bottom dishes. Nikon sells another oil that works with
> this type of dishes but it is very toxic so I am not so happy to use it.
> The oil that Zeiss sells however is compatible with plastic bottom dishes
> and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a
> different manufacturers use different type of rubber seal to assemble their
> lenses and the seal might not be compatible with just any oil. Has anyone
> tried the Zeiss oil on Nikon objectives?
>
> As always, thanks to everyone for your help
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: [phone]
> office: [phone]
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>
>

--
Best,

Gary Laevsky, Ph.D.
Confocal Imaging Facility Manager
Dept. of Molecular Biology
Washington Rd.
Princeton University
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310

[George McNamara]

*****
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 Sylvie,

Does your standard Nikon oil melt the COP (cyclic olefin polymer) Aurora
Microplates?

http://www.auroramicroplates.com/

I did not see refractive index on their applications page

http://www.auroramicroplates.com/application.html

Yes, COP is not glass, but you gave not gotten glass bottom plates that
work. Get An Aurora plate(s), fill the middle wells with oil, put the
plate on a paper towel(s), and leave it for the weekend.

Skirt vs edge wells - Aurora developed the first 3456 well plate (and
may have invented the 1536 well plate), so if the Aurora 96 or 384 well
plates all have skirts, go to 1536 or 3456 and live without the outer wells.

With respect to using Zeiss oil on your Nikon objective lens on your
current workflow, test 'instant gratification' (by GPU) deconvolution
from both www.microvolution.com and SVI Huygens (I haven't checked to
see if any other deconvolution vendor has added 'instant
gratification'). You should be able to arrange with each company to send
them your current data to evaluate. Yes, deconvolution will improve
confocal images -- may even fix your R.I. mismatch issue.


George
p.s. disclosure: the images in the middle of the microvolution.com home
page are from our lab (widefield with Leica 63x/1.4NA lens using Leica oil).

On 2/16/2016 4:46 AM, Sylvie Le Guyader 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 everyone on the list
>
> I have 2 questions:
>
> 1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
> Here are our requirements:
> - 96 well plates
> - borosilicate bottom
> - thickness #1.5 (170 or 175 um)
> - no skirt as it makes it impossible to image the outer wells with wide objectives
> - flat (ie no slant of a full field of view with a 10x objective)
>
> None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!
>
> Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?
>
>
> 2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?
>
> As always, thanks to everyone for your help
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: [phone]
> office: [phone]
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>
>

--



George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

[Sylvie Le Guyader]

*****
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*****

Hi Eric

Actually the Matrical plates are those who gave us a lot of trouble. Everything was fine for years then suddenly the cells were dying in about 30% of the wells. After extensive troubleshooting with the company and still no idea what was going wrong, we have started a long and painful search for new plates.
Hence my email. I am now collecting the brand of plates we have tried which are slanted. As far as I can tell (feedback from users), only the Matrical plates were toxic. However most plates are slanted.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Eric Griffis (Staff)
Sent: den 16 februari 2016 13:31
To: [hidden email]
Subject: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
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 Sylvie,

We've had good success with plates from Matrical (which may now be sold as Brooks?). The wells are square, so you have more surface area, and I've found that they have less toxicity compared to some plates from other vendors, which were more variable. They will also usually send you a sample plate to test in your application.

Hope this is helpful.

Best regards,

Eric



Centre for Gene Regulation and Expression College of Life Sciences University of Dundee MSI/WTB/JBC Complex Dow Street Dundee DD1 5EH United KIngdom

[phone]
[hidden email]<mailto:[hidden email]>
http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html



On 16 Feb 2016, at 10:46, Sylvie Le Guyader <[hidden email]<mailto:[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 everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>


The University of Dundee is a registered Scottish Charity, No: SC015096

[Sylvie Le Guyader]

*****
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 George

Thanks for suggesting Aurora plates. We have not tried them so I will request a sample to test.
At least when it comes to Ibidi optical bottom, the Nikon oil doesn't take more than a few min to destroy the plastic. It doesn't melt holes into it but it makes it opaque.

Concerning using oil from one brand onto objectives of another brand, my concern was more about chemically damaging the objective than getting crap RI mismatch although I guess that would have popped up next on my list of concerns. :)

The different reaction one gets on plastic bottom plates with different oils clearly shows that their chemistry is very different. I am concerned with damaging the lens ring.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: den 16 februari 2016 14:41
To: [hidden email]
Subject: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
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 Sylvie,

Does your standard Nikon oil melt the COP (cyclic olefin polymer) Aurora Microplates?

http://www.auroramicroplates.com/

I did not see refractive index on their applications page

http://www.auroramicroplates.com/application.html

Yes, COP is not glass, but you gave not gotten glass bottom plates that work. Get An Aurora plate(s), fill the middle wells with oil, put the plate on a paper towel(s), and leave it for the weekend.

Skirt vs edge wells - Aurora developed the first 3456 well plate (and may have invented the 1536 well plate), so if the Aurora 96 or 384 well plates all have skirts, go to 1536 or 3456 and live without the outer wells.

With respect to using Zeiss oil on your Nikon objective lens on your current workflow, test 'instant gratification' (by GPU) deconvolution from both www.microvolution.com and SVI Huygens (I haven't checked to see if any other deconvolution vendor has added 'instant gratification'). You should be able to arrange with each company to send them your current data to evaluate. Yes, deconvolution will improve confocal images -- may even fix your R.I. mismatch issue.


George
p.s. disclosure: the images in the middle of the microvolution.com home page are from our lab (widefield with Leica 63x/1.4NA lens using Leica oil).

On 2/16/2016 4:46 AM, Sylvie Le Guyader 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 everyone on the list
>
> I have 2 questions:
>
> 1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
> Here are our requirements:
> - 96 well plates
> - borosilicate bottom
> - thickness #1.5 (170 or 175 um)
> - no skirt as it makes it impossible to image the outer wells with
> wide objectives
> - flat (ie no slant of a full field of view with a 10x objective)
>
> None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!
>
> Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?
>
>
> 2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?
>
> As always, thanks to everyone for your help
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: [phone]
> office: [phone]
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>
>

--



George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab) University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

[Shinogle, Heather]

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Sylvie,

I have had good success with plates from Cellvis, no issues with the size of plate with my Prior stage or issues with autofocus (we have an Olympus microscope equipped with a ZDC).  They will also provide you with a sample to test.

https://www.cellvis.com/_96-well-glass-bottom-plates_/products_by_category.php?cat_id=11

Sincerely,

Heather



Heather Shinogle
Research Lab Director
Microscopy and Analytical Imaging Core
Lab - 1043 Haworth
Office - 1036 Haworth
785-864-4380
[hidden email]<mailto:[hidden email]>




On Feb 16, 2016, at 4:46 AM, Sylvie Le Guyader <[hidden email]<mailto:[hidden email]>> wrote:

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Dear everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Sylvie Le Guyader]

*****
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Following an offline email by Eric who suggested the use of MatTek MW plates, I would like all the vendors out there to realize that companies that offer the chance to test their products for free (like Ibidi for example but unlike MatTek with their MW plates) have a very strong head start compared to those who don't.

Considering that many MW plates either kill cells or are not flat at all, one doesn't want to invest into buying a whole box of them just to test the quality and often be disappointed. I would like to suggest to all vendors that if you believe your product is of excellent quality, you prove it by offering to send 1 free MW plate per lab. :)

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Sylvie Le Guyader
Sent: den 16 februari 2016 14:53
To: 'Confocal Microscopy List'
Subject: RE: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

Hi Eric

Actually the Matrical plates are those who gave us a lot of trouble. Everything was fine for years then suddenly the cells were dying in about 30% of the wells. After extensive troubleshooting with the company and still no idea what was going wrong, we have started a long and painful search for new plates.
Hence my email. I am now collecting the brand of plates we have tried which are slanted. As far as I can tell (feedback from users), only the Matrical plates were toxic. However most plates are slanted.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Eric Griffis (Staff)
Sent: den 16 februari 2016 13:31
To: [hidden email]
Subject: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

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Hi Sylvie,

We've had good success with plates from Matrical (which may now be sold as Brooks?). The wells are square, so you have more surface area, and I've found that they have less toxicity compared to some plates from other vendors, which were more variable. They will also usually send you a sample plate to test in your application.

Hope this is helpful.

Best regards,

Eric



Centre for Gene Regulation and Expression College of Life Sciences University of Dundee MSI/WTB/JBC Complex Dow Street Dundee DD1 5EH United KIngdom

[phone]
[hidden email]<mailto:[hidden email]>
http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html



On 16 Feb 2016, at 10:46, Sylvie Le Guyader <[hidden email]<mailto:[hidden email]>>
 wrote:

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*****

Dear everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>


The University of Dundee is a registered Scottish Charity, No: SC015096

[Guillermo Marques]

*****
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Sylvie,

We have successfully used Nikon NF oil in the past with Ibidi dishes. Is that the oil you tested that is toxic?

Guillermo

Guillermo Marqués
University Imaging Centers
University of Minnesota
Jackson Hall 1-151
321 Church St SE
Minneapolis, MN55455

> On Feb 16, 2016, at 4:46 AM, Sylvie Le Guyader <[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 everyone on the list
>
> I have 2 questions:
>
> 1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
> Here are our requirements:
> - 96 well plates
> - borosilicate bottom
> - thickness #1.5 (170 or 175 um)
> - no skirt as it makes it impossible to image the outer wells with wide objectives
> - flat (ie no slant of a full field of view with a 10x objective)
>
> None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!
>
> Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?
>
>
> 2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?
>
> As always, thanks to everyone for your help
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: [phone]
> office: [phone]
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Sylvie Le Guyader]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

The oil we use routinely and that is provided by Nikon is MXA20235. It is not toxic but we cannot use it with plastic bottom dishes.
The oil that doesn't dissolve optical plastic bottoms is NF2 (MXA22126). That is very toxic according to the MSDA. :(

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guillermo Marques
Sent: den 16 februari 2016 16:17
To: [hidden email]
Subject: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Sylvie,

We have successfully used Nikon NF oil in the past with Ibidi dishes. Is that the oil you tested that is toxic?

Guillermo

Guillermo Marqués
University Imaging Centers
University of Minnesota
Jackson Hall 1-151
321 Church St SE
Minneapolis, MN55455

> On Feb 16, 2016, at 4:46 AM, Sylvie Le Guyader <[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 everyone on the list
>
> I have 2 questions:
>
> 1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
> Here are our requirements:
> - 96 well plates
> - borosilicate bottom
> - thickness #1.5 (170 or 175 um)
> - no skirt as it makes it impossible to image the outer wells with wide objectives
> - flat (ie no slant of a full field of view with a 10x objective)
>
> None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!
>
> Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?
>
>
> 2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?
>
> As always, thanks to everyone for your help
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: [phone]
> office: [phone]
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Sylvie Le Guyader]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Heather

Thanks for the suggestion. These plates have a skirt but it is only 2.5 mm so that might work. I will contact them to request a sample. :)

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Shinogle, Heather
Sent: den 16 februari 2016 15:54
To: [hidden email]
Subject: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Sylvie,

I have had good success with plates from Cellvis, no issues with the size of plate with my Prior stage or issues with autofocus (we have an Olympus microscope equipped with a ZDC).  They will also provide you with a sample to test.

https://www.cellvis.com/_96-well-glass-bottom-plates_/products_by_category.php?cat_id=11

Sincerely,

Heather



Heather Shinogle
Research Lab Director
Microscopy and Analytical Imaging Core
Lab - 1043 Haworth
Office - 1036 Haworth
785-864-4380
[hidden email]<mailto:[hidden email]>




On Feb 16, 2016, at 4:46 AM, Sylvie Le Guyader <[hidden email]<mailto:[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 everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Dan Focht]

*****
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Sylvie

Multiwell plates are injection molded at high temperature then partially cooled while still in the mold.
After partial cooling they are ejected and left to continue cooling.
This results in strain in the plastic that continues to affect the flatness of the surface where glass is attached at the bottom of the well.
Once the glass is attached, usually by an adhesive, the glass tends to conform to the plastic.
This may explain some of the issues you are having.
Although great effort goes into making sure the bottom of the plate is flat and parallel with the mounting surface, it is still an injection molded part and subject to inherent distortions.
If you are working with mammalian cells and warming the plates during microscopy the heat propagation adds a whole new wrinkle to your problem.

The smaller 8 well coverglass bottomed chambers such as Ibidi, LabTek 1 and 2 are more geometrically stable and can be warmed without introducing Z axis drift in A Bioptechs Stable Z warmer.
I don't know if your protocol requires 96 wells but if you can use 8 wells at a time you will have better thermal and optical results.


Dan



On Feb 16, 2016, at 9:12 AM, Sylvie Le Guyader wrote:

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Dear George

Thanks for suggesting Aurora plates. We have not tried them so I will request a sample to test.
At least when it comes to Ibidi optical bottom, the Nikon oil doesn't take more than a few min to destroy the plastic. It doesn't melt holes into it but it makes it opaque.

Concerning using oil from one brand onto objectives of another brand, my concern was more about chemically damaging the objective than getting crap RI mismatch although I guess that would have popped up next on my list of concerns. :)

The different reaction one gets on plastic bottom plates with different oils clearly shows that their chemistry is very different. I am concerned with damaging the lens ring.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: den 16 februari 2016 14:41
To: [hidden email]
Subject: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

*****
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*****

Hi Sylvie,

Does your standard Nikon oil melt the COP (cyclic olefin polymer) Aurora Microplates?

http://www.auroramicroplates.com/

I did not see refractive index on their applications page

http://www.auroramicroplates.com/application.html

Yes, COP is not glass, but you gave not gotten glass bottom plates that work. Get An Aurora plate(s), fill the middle wells with oil, put the plate on a paper towel(s), and leave it for the weekend.

Skirt vs edge wells - Aurora developed the first 3456 well plate (and may have invented the 1536 well plate), so if the Aurora 96 or 384 well plates all have skirts, go to 1536 or 3456 and live without the outer wells.

With respect to using Zeiss oil on your Nikon objective lens on your current workflow, test 'instant gratification' (by GPU) deconvolution from both www.microvolution.com and SVI Huygens (I haven't checked to see if any other deconvolution vendor has added 'instant gratification'). You should be able to arrange with each company to send them your current data to evaluate. Yes, deconvolution will improve confocal images -- may even fix your R.I. mismatch issue.


George
p.s. disclosure: the images in the middle of the microvolution.com home page are from our lab (widefield with Leica 63x/1.4NA lens using Leica oil).

On 2/16/2016 4:46 AM, Sylvie Le Guyader 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 everyone on the list
>
> I have 2 questions:
>
> 1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
> Here are our requirements:
> - 96 well plates
> - borosilicate bottom
> - thickness #1.5 (170 or 175 um)
> - no skirt as it makes it impossible to image the outer wells with
> wide objectives
> - flat (ie no slant of a full field of view with a 10x objective)
>
> None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!
>
> Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?
>
>
> 2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?
>
> As always, thanks to everyone for your help
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: [phone]
> office: [phone]
> LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>
>

--



George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab) University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

Dan Focht
Bioptechs, Inc.
3560 Beck Rd.
Butler, PA 16002
www.bioptechs.com
P: [phone]
F: [phone]
[hidden email]

[Ann-W]

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Hi Silvie

Similarly to Gabor we use the same oil for most of the objectives on our systems. The only exception is the super-resolution microscopes where we do use the manufacturers recommendation.

Wrt plates I can only agree, it's difficult to get hold of reproducibly good 96 well plates. Iwaki plates, pre tsumani, were very good. Others may be able to advise if they are available again now. At the moment I'd suggest Mat-tek or Greiner's glass bottomed multiwell plates if your budget can stretch to it. I also found switching objectives on my Zeiss system to the older and slimmer type worked well for acquiring the difficult to visualise edge wells in a 96 well plate. The new objectives for most systems are a bit wider and conflict with the edge of the plate and more importantly the multiwell plate holder stage insert.

We found that the choice of plate holder for imaging multiwell plates made a lot of difference too. If you can get one with two springs on either side inside the plate holder this might help so the tension across the plate is identical. The ones which hold the plate from the corner in our experience didn't work very well. They put pressure on the plate and made it sit at a very slight slant. So the plates were actually fairly flat but the way they were sitting in the holder made them less so.

You'll need a good autofocusing algorithm as well, we're in the process of going through the algorithms in Micromanager and have found a couple of them work well for us. Metamorph was pretty good but I can't comment on others.

All the best

Ann


Dr Ann Wheeler
Advanced Imaging Resource, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Csúcs Gábor
Sent: 16 February 2016 12:14
To: [hidden email]
Subject: Re: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

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Dear Sylvie,

I will need to check on the plate issue, but for the oils: we have been always using Zeiss oil on all of our microscopes (Zeiss, Leica, Olympus, Nikon). So far (over a couple of years) with no observable effect.
Concerning the plates: perhaps you could share your list of plates that you have tried already (without success).

Greetings    Gabor



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Dienstag, 16. Februar 2016 11:47
To: [hidden email]
Subject: flat glass bottom multiwell plates and Zeiss oil on Nikon objectives

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Dear everyone on the list

I have 2 questions:

1-      It proves surprisingly difficult to find multiwell plates that work well for our microscopy purpose which is high res microscopy on multiple wells.
Here are our requirements:
- 96 well plates
- borosilicate bottom
- thickness #1.5 (170 or 175 um)
- no skirt as it makes it impossible to image the outer wells with wide objectives
- flat (ie no slant of a full field of view with a 10x objective)

None of the plates we tried are actually flat. The glass is good but the plastic part is so slanted or distorted that we see the slope even in a single field of view so using a hardware autofocus doesn't help. Many plates have a glass bottom of thickness 145 um which is the wrong specification for the objectives. Some plates leak. Some kill the cells in about 30% of the wells!

Could people on the list and vendors recommend glass bottom plates that fit with the requirements above?


2-      My second question is related. We have many Nikon microscopes. The oil Nikon sells for their objectives melts the plastic used in many 'optical' plastic bottom dishes. Nikon sells another oil that works with this type of dishes but it is very toxic so I am not so happy to use it. The oil that Zeiss sells however is compatible with plastic bottom dishes and is not as toxic as the Nikon oil. But I am a bit hesitant to use it a different manufacturers use different type of rubber seal to assemble their lenses and the seal might not be compatible with just any oil. Has anyone tried the Zeiss oil on Nikon objectives?

As always, thanks to everyone for your help

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website<http://ki.se/en/bionut/welcome-to-the-lci-unit>

[Jeremy Adler-4]

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Is there a similar alignment problem with glass bottomed petri dishes

 - is the glass perfectly aligned with the imaging plane when the petri dish rests on its base ?

Re Ibidi - their website lists oils are OK with their plastic, perhaps they could also list those that must be avoided.

Jeremy Adler
BioVis
Uppsala Universitet


[phone]

http://www.biovis.uu.se