Permanox chamber slides issues

[daj1u06]

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Hi, we have a user who has grown all his cells inNunc labtek  permanox chamber slides, pealed the chambers off and mounted the plastic slides with cells under coverslips. We are having huge problems imaging them because of enormous Z drift - you set up the Z stack and when you start collecting the series the values are already way off by up to 10um. It is almost as if the plastic slides are reacting to the laser and swelling and bowing up away from the objective (inverted SP8 system). We have tried holding the slide down with glass slides but still real issues.

Has anyone experienced similar issues / has any ideas. Thanks in advance,

Dave Johnston, Biomedical Imaging Unit, Southampton.

[TSwayne]

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Hi Dave,

Is this happening under oil immersion? If so, I’ve struggled with the same thing and yes, a glass slide helps but it’s not perfect. It’s worse if the preparation is too thick and you’re approaching the working distance of the objective.  Leftover glue from the chambers, and excess mounting media, are the usual culprits.

For finicky cells that “don’t like glass,” we've had good luck with ibidi polymer coverslip chambers like this:
https://ibidi.com/chambered-coverslips/13--slide-8-well-ibitreat.html  (no affiliation). The wells are in a rigid frame and we have not noticed any stability issues with z series.

(As a bonus, the wells are intelligently placed in the center of the slide, not all the way at the end, so you can access all 8 wells from a wider variety of stage inserts.)

You can’t remove the chambers, but you can fix and stain in the wells, and that seems to work for our users who have tried it.

Hope this helps.
Theresa

------------------------------------
Theresa Swayne, Ph.D.
Associate Research Scientist
Manager, Confocal and Specialized Microscopy Shared Resource<http://hiccc.columbia.edu/research/sharedresources/confocal>

Herbert Irving Comprehensive Cancer Center
Columbia University Irving Medical Center
1130 St. Nicholas Ave., Room 222A
New York, NY 10032
Phone: [phone]
[hidden email]<mailto:[hidden email]>



From: Confocal Microscopy List <[hidden email]> on behalf of Dave Johnston <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Tuesday, May 7, 2019 at 3:57 AM
To: "[hidden email]" <[hidden email]>
Subject: Permanox chamber slides issues

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Hi, we have a user who has grown all his cells inNunc labtek  permanox chamber slides, pealed the chambers off and mounted the plastic slides with cells under coverslips. We are having huge problems imaging them because of enormous Z drift - you set up the Z stack and when you start collecting the series the values are already way off by up to 10um. It is almost as if the plastic slides are reacting to the laser and swelling and bowing up away from the objective (inverted SP8 system). We have tried holding the slide down with glass slides but still real issues.

Has anyone experienced similar issues / has any ideas. Thanks in advance,

Dave Johnston, Biomedical Imaging Unit, Southampton.

[Cammer, Michael]

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They warp and swell.  Non-aqueous mounting media may cause the plastic to swell and irreversably warp.
  If we have to use them, we back them with a glass slide and clip tight.  This is not a guarranteed solution, but usually works.


Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
[hidden email]  http://nyulmc.org/micros  http://microscopynotes.com/
Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567

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From: Confocal Microscopy List <[hidden email]> on behalf of Swayne, Theresa C. <[hidden email]>
Sent: Tuesday, May 7, 2019 9:17:00 AM
To: [hidden email]
Subject: Re: Permanox chamber slides issues

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Hi Dave,

Is this happening under oil immersion? If so, I’ve struggled with the same thing and yes, a glass slide helps but it’s not perfect. It’s worse if the preparation is too thick and you’re approaching the working distance of the objective.  Leftover glue from the chambers, and excess mounting media, are the usual culprits.

For finicky cells that “don’t like glass,” we've had good luck with ibidi polymer coverslip chambers like this:
https://urldefense.proofpoint.com/v2/url?u=https-3A__ibidi.com_chambered-2Dcoverslips_13-2D-2Dslide-2D8-2Dwell-2Dibitreat.html&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=g3XLqVBcPQO0UZPw6h9YBhDHq7ubeMkOzKhQmm-ITMg&s=PoNmW99GaktRrdbIj1hP3bdOwUVzxTvxd6o7pUnrxxo&e=  (no affiliation). The wells are in a rigid frame and we have not noticed any stability issues with z series.

(As a bonus, the wells are intelligently placed in the center of the slide, not all the way at the end, so you can access all 8 wells from a wider variety of stage inserts.)

You can’t remove the chambers, but you can fix and stain in the wells, and that seems to work for our users who have tried it.

Hope this helps.
Theresa

------------------------------------
Theresa Swayne, Ph.D.
Associate Research Scientist
Manager, Confocal and Specialized Microscopy Shared Resource<https://urldefense.proofpoint.com/v2/url?u=http-3A__hiccc.columbia.edu_research_sharedresources_confocal&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=g3XLqVBcPQO0UZPw6h9YBhDHq7ubeMkOzKhQmm-ITMg&s=jafTGTsFw6U6cQBtz7otT46ul5tYArcsHSkIWG4LG7c&e=>

Herbert Irving Comprehensive Cancer Center
Columbia University Irving Medical Center
1130 St. Nicholas Ave., Room 222A
New York, NY 10032
Phone: [phone]
[hidden email]<mailto:[hidden email]>



From: Confocal Microscopy List <[hidden email]> on behalf of Dave Johnston <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Tuesday, May 7, 2019 at 3:57 AM
To: "[hidden email]" <[hidden email]>
Subject: Permanox chamber slides issues

*****
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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=G2MiLlal7SXE3PeSnG8W6_JBU6FcdVjSsBSbw6gcR0U&r=JzqbFzGMeaMAlwu5KvYtC8ig-FF8xbvhI6BlFtbXky8&m=RJ_Zg7WjVX8dVyWEY0TAyLWEH97MAsAVyIahwvV6eNM&s=NLYpobqwbPQpMHprzObL922OmmrIcVpw-k8gliZ6DOw&e= and include the link in your posting.
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Hi, we have a user who has grown all his cells inNunc labtek  permanox chamber slides, pealed the chambers off and mounted the plastic slides with cells under coverslips. We are having huge problems imaging them because of enormous Z drift - you set up the Z stack and when you start collecting the series the values are already way off by up to 10um. It is almost as if the plastic slides are reacting to the laser and swelling and bowing up away from the objective (inverted SP8 system). We have tried holding the slide down with glass slides but still real issues.

Has anyone experienced similar issues / has any ideas. Thanks in advance,

Dave Johnston, Biomedical Imaging Unit, Southampton.


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[Jacqueline Ross]

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Hi Dave,

We've had similar issues with these on our upright confocal but it's easier there as you can put a slide underneath it to support it. They are just too bendy.

Haven't had these come to the invert (not sure why) but like Theresa, we usually use the ibidi 8 well or similar chambers there. I would suggest what you have already tried, putting another slide on the back to add extra weight.

Cheers,

Jacqui

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dave Johnston
Sent: Tuesday, 7 May 2019 7:57 p.m.
To: [hidden email]
Subject: Permanox chamber slides issues

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Hi, we have a user who has grown all his cells inNunc labtek  permanox chamber slides, pealed the chambers off and mounted the plastic slides with cells under coverslips. We are having huge problems imaging them because of enormous Z drift - you set up the Z stack and when you start collecting the series the values are already way off by up to 10um. It is almost as if the plastic slides are reacting to the laser and swelling and bowing up away from the objective (inverted SP8 system). We have tried holding the slide down with glass slides but still real issues.

Has anyone experienced similar issues / has any ideas. Thanks in advance,

Dave Johnston, Biomedical Imaging Unit, Southampton.

[daj1u06]

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Thanks everyone for your suggestions, on and off board. Yes we are using glycerol immersion.

Unfortunately, this was a student project and they set up the experiments under supervisor instruction without either of them having consuled us beforehand and only came to us at the end of a long project for the imaging. We would have advised Ibidi chamberslides or coverslip cultures to mount on slides as our standard protocol from the outset had they asked, but it was too late!

Cheers,

DAJ