Biological inert coverslip sealing

[Antonio Jose Pereira]

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Hi all,

For some purposes in live cell imaging we assemble a chamber with a coverslip on top of a slide.
We use two halves of another coverslip to act as spacers.

The  issue is the sealing. We've been using VALAP (a kind of wax) that we  melt and then use to seal all the way through the periphery of the  coverslip.

It works, but it would be more convenient to have something that does not have to be heated, and that would harden just by  contact with air.
A bit like the nail varnish used for fixed material ... but here we need something which is biologically inert.

Any suggestions? Thanks a lot.

Antonio



António Pereira
Chromosome Instability and Dynamics Group, Maiato lab

IBMC - Institute for Molecular and Cell Biology
Porto- Portugal


António Pereira
IBMC - Instituto de Biologia Molecular e Celular
Grupo Instabilidade e Dinâmica Cromossómica

Rua do Campo Alegre, 823
4150-180 Porto
Portugal

[phone] (ext# 1620)
[hidden email]

[Josef Gotzmann]

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Dear Antonio,

For this purpose we use dental cement (Picodent TwinSil 22;
http://www.picodent.de/), which is not only biologically inert, though also
can be easily removed after imaging to save the sample for post-imaging
processing.

Josef


On Thu, 27 Feb 2014 15:52:37 +0000, Antonio J Pereira <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>
>Hi all,
>
>For some purposes in live cell imaging we assemble a chamber with a
coverslip on top of a slide.
>We use two halves of another coverslip to act as spacers.
>
>The  issue is the sealing. We've been using VALAP (a kind of wax) that we
melt and then use to seal all the way through the periphery of the  coverslip.
>
>It works, but it would be more convenient to have something that does not
have to be heated, and that would harden just by  contact with air.
>A bit like the nail varnish used for fixed material ... but here we need
something which is biologically inert.

>
>Any suggestions? Thanks a lot.
>
>Antonio
>
>
>
>António Pereira
>Chromosome Instability and Dynamics Group, Maiato lab
>
>IBMC - Institute for Molecular and Cell Biology
>Porto- Portugal
>
>
>António Pereira
>IBMC - Instituto de Biologia Molecular e Celular
>Grupo Instabilidade e Dinâmica Cromossómica
>
>Rua do Campo Alegre, 823
>4150-180 Porto
>Portugal
>
>[phone] (ext# 1620)
>[hidden email]

[mmodel]

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We use silicone grease

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Antonio J Pereira
Sent: Thursday, February 27, 2014 10:53 AM
To: [hidden email]
Subject: Biological inert coverslip sealing

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Hi all,

For some purposes in live cell imaging we assemble a chamber with a coverslip on top of a slide.
We use two halves of another coverslip to act as spacers.

The  issue is the sealing. We've been using VALAP (a kind of wax) that we  melt and then use to seal all the way through the periphery of the  coverslip.

It works, but it would be more convenient to have something that does not have to be heated, and that would harden just by  contact with air.
A bit like the nail varnish used for fixed material ... but here we need something which is biologically inert.

Any suggestions? Thanks a lot.

Antonio



António Pereira
Chromosome Instability and Dynamics Group, Maiato lab

IBMC - Institute for Molecular and Cell Biology
Porto- Portugal


António Pereira
IBMC - Instituto de Biologia Molecular e Celular Grupo Instabilidade e Dinâmica Cromossómica

Rua do Campo Alegre, 823
4150-180 Porto
Portugal

[phone] (ext# 1620)
[hidden email]

[Oshel, Philip Eugene]

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Do you have access to Canada Balsam?
Used extensively for mounting microarthropods like mites, etc. (Or it
was, anyway.

Phil

On 02/27/2014 10:52 , Antonio J Pereira wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Hi all,
>
> For some purposes in live cell imaging we assemble a chamber with a coverslip on top of a slide.
> We use two halves of another coverslip to act as spacers.
>
> The  issue is the sealing. We've been using VALAP (a kind of wax) that we  melt and then use to seal all the way through the periphery of the  coverslip.
>
> It works, but it would be more convenient to have something that does not have to be heated, and that would harden just by  contact with air.
> A bit like the nail varnish used for fixed material ... but here we need something which is biologically inert.
>
> Any suggestions? Thanks a lot.
>
> Antonio
>
>
>
> António Pereira
> Chromosome Instability and Dynamics Group, Maiato lab
>
> IBMC - Institute for Molecular and Cell Biology
> Porto- Portugal
>
>
> António Pereira
> IBMC - Instituto de Biologia Molecular e Celular
> Grupo Instabilidade e Dinâmica Cromossómica
>
> Rua do Campo Alegre, 823
> 4150-180 Porto
> Portugal
>
> [phone] (ext# 1620)
> [hidden email]
>

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
[phone]

[Littlejohn, George]

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Dear Antonio,

I can recommend Carolina Observation Gel (polydimethylsiloxane, and supplied by Blades Biological in the UK).

All the best,
George

******************************
Dr. George Littlejohn
Halpin Laboratory,
School of Biosciences,
University of Exeter,
Geoffrey Pope Building,
Stocker Road,
EX4 4QD, UK

Telephone: [phone]
E-mail:  [hidden email]<mailto:[hidden email]>

http://projects.exeter.ac.uk/ein/
https://twitter.com/geolittlejohn


On 27 Feb 2014, at 16:05, "Antonio J Pereira" <[hidden email]<mailto:[hidden email]>> wrote:

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Hi all,

For some purposes in live cell imaging we assemble a chamber with a coverslip on top of a slide.
We use two halves of another coverslip to act as spacers.

The  issue is the sealing. We've been using VALAP (a kind of wax) that we  melt and then use to seal all the way through the periphery of the  coverslip.

It works, but it would be more convenient to have something that does not have to be heated, and that would harden just by  contact with air.
A bit like the nail varnish used for fixed material ... but here we need something which is biologically inert.

Any suggestions? Thanks a lot.

Antonio



António Pereira
Chromosome Instability and Dynamics Group, Maiato lab

IBMC - Institute for Molecular and Cell Biology
Porto- Portugal


António Pereira
IBMC - Instituto de Biologia Molecular e Celular
Grupo Instabilidade e Dinâmica Cromossómica

Rua do Campo Alegre, 823
4150-180 Porto
Portugal

[phone] (ext# 1620)
[hidden email]<mailto:[hidden email]>

[Julio Vazquez]

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Don't know if this will work for your purpose, but I used to make a ring (or square) with nail polish on the slide. let it dry and apply more layers to increase the thickness as needed. I let the nail polish dry out a couple of hours at room temperature (or faster in a 37oC oven) to evaporate solvents. This will make it life- and GFP-compatible. Put your specimen inside the well and add buffer/medium. Put a coverslip on top. The coverslip will typically stick to the nail polish, which serves both as a spacer and sealant.

Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109

http://www.fhcrc.org/en.html

--



On Feb 27, 2014, at 7:52 AM, Antonio J Pereira wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Hi all,
>
> For some purposes in live cell imaging we assemble a chamber with a coverslip on top of a slide.
> We use two halves of another coverslip to act as spacers.
>
> The  issue is the sealing. We've been using VALAP (a kind of wax) that we  melt and then use to seal all the way through the periphery of the  coverslip.
>
> It works, but it would be more convenient to have something that does not have to be heated, and that would harden just by  contact with air.
> A bit like the nail varnish used for fixed material ... but here we need something which is biologically inert.
>
> Any suggestions? Thanks a lot.
>
> Antonio
>
>
>
> António Pereira
> Chromosome Instability and Dynamics Group, Maiato lab
>
> IBMC - Institute for Molecular and Cell Biology
> Porto- Portugal
>
>
> António Pereira
> IBMC - Instituto de Biologia Molecular e Celular
> Grupo Instabilidade e Dinâmica Cromossómica
>
> Rua do Campo Alegre, 823
> 4150-180 Porto
> Portugal
>
> [phone] (ext# 1620)
> [hidden email]

[lechristophe]

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

We have been using dental cement on fixed samples, and as stated it should
be OK with live samples.

For live samples I prefer using EMS Diasum silicone hybridization chamber:
they exist in a lot different sizes and shapes to accommodate pretty much
any type of coverslip. Some of them have an adhesive side that sticks on
the slide, then you just need to fill the well (a few 10s-100s of µL will
do) and gently press the coverslip, then aspirate the excess medium to seal
the chamber

It is easy to remove the coverslip for further processing, and the
slide/chamber assembly is reusable. If you worry about the coverslip moving
during imaging, we have been doing single particle tracking and
super-resolution microscopy using these chambers without any noticeable
problems compared to dental cement.

Check here:
http://www.emsdiasum.com/microscopy/products/preparation/hybridization.aspx

For our 18mm round coverslips we use the one-side adhesive, 13mm wide,
0.6mm deep ref. 70-334-A:
http://www.emsdiasum.com/microscopy/products/preparation/hybridization.aspx#70334

Hope this helps,

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France




2014-02-27 16:52 GMT+01:00 Antonio J Pereira <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Hi all,
>
> For some purposes in live cell imaging we assemble a chamber with a
> coverslip on top of a slide.
> We use two halves of another coverslip to act as spacers.
>
> The  issue is the sealing. We've been using VALAP (a kind of wax) that we
>  melt and then use to seal all the way through the periphery of the
>  coverslip.
>
> It works, but it would be more convenient to have something that does not
> have to be heated, and that would harden just by  contact with air.
> A bit like the nail varnish used for fixed material ... but here we need
> something which is biologically inert.
>
> Any suggestions? Thanks a lot.
>
> Antonio
>
>
>
> António Pereira
> Chromosome Instability and Dynamics Group, Maiato lab
>
> IBMC - Institute for Molecular and Cell Biology
> Porto- Portugal
>
>
> António Pereira
> IBMC - Instituto de Biologia Molecular e Celular
> Grupo Instabilidade e Dinâmica Cromossómica
>
> Rua do Campo Alegre, 823
> 4150-180 Porto
> Portugal
>
> [phone] (ext# 1620)
> [hidden email]
>

[Antonio Jose Pereira]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thank you all for the suggestions.
I have now a few things to try.



António Pereira
Chromosome Instability and Dynamics Group, Maiato lab
IBMC - Institute for Molecular and Cell Biology
Porto- Portugal


-----Confocal Microscopy List <[hidden email]> wrote: -----
To: [hidden email]
From: Christophe Leterrier
Sent by: Confocal Microscopy List
Date: 02/27/2014 08:50PM
Subject: Re: Biological inert coverslip sealing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Hi Antonio,

We have been using dental cement on fixed samples, and as stated it should
be OK with live samples.

For live samples I prefer using EMS Diasum silicone hybridization chamber:
they exist in a lot different sizes and shapes to accommodate pretty much
any type of coverslip. Some of them have an adhesive side that sticks on
the slide, then you just need to fill the well (a few 10s-100s of µL will
do) and gently press the coverslip, then aspirate the excess medium to seal
the chamber

It is easy to remove the coverslip for further processing, and the
slide/chamber assembly is reusable. If you worry about the coverslip moving
during imaging, we have been doing single particle tracking and
super-resolution microscopy using these chambers without any noticeable
problems compared to dental cement.

Check here:
http://www.emsdiasum.com/microscopy/products/preparation/hybridization.aspx

For our 18mm round coverslips we use the one-side adhesive, 13mm wide,
0.6mm deep ref. 70-334-A:
http://www.emsdiasum.com/microscopy/products/preparation/hybridization.aspx#70334

Hope this helps,

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France




2014-02-27 16:52 GMT+01:00 Antonio J Pereira <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Hi all,
>
> For some purposes in live cell imaging we assemble a chamber with a
> coverslip on top of a slide.
> We use two halves of another coverslip to act as spacers.
>
> The  issue is the sealing. We've been using VALAP (a kind of wax) that we
>  melt and then use to seal all the way through the periphery of the
>  coverslip.
>
> It works, but it would be more convenient to have something that does not
> have to be heated, and that would harden just by  contact with air.
> A bit like the nail varnish used for fixed material ... but here we need
> something which is biologically inert.
>
> Any suggestions? Thanks a lot.
>
> Antonio
>
>
>
> António Pereira
> Chromosome Instability and Dynamics Group, Maiato lab
>
> IBMC - Institute for Molecular and Cell Biology
> Porto- Portugal
>
>
> António Pereira
> IBMC - Instituto de Biologia Molecular e Celular
> Grupo Instabilidade e Dinâmica Cromossómica
>
> Rua do Campo Alegre, 823
> 4150-180 Porto
> Portugal
>
> [phone] (ext# 1620)
> [hidden email]
>

[phil laissue-2]

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To join, leave or search the confocal microscopy listserv, go to:
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In addition to dental cement, I've been using rubber cement, which works
well and is cheap ('Fixogum' from Marabu with a pipette tip - no commercial
interest).
I generally advise against nail varnish, due to its strong autofluorescence
etc:

Fixation, mounting and sealing with nail polish of cell specimens lead to
incorrect FRET measurements using acceptor photobleaching.
Rodighiero S, Bazzini C, Ritter M, Fürst J, Botta G, Meyer G, Paulmichl M.
Cell Physiol Biochem. 2008;21(5-6):489-98. doi: 10.1159/000129642. Epub
2008 Apr 24.

_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue


On 27 February 2014 20:42, Christophe Leterrier <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Antonio,
>
> We have been using dental cement on fixed samples, and as stated it should
> be OK with live samples.
>
> For live samples I prefer using EMS Diasum silicone hybridization chamber:
> they exist in a lot different sizes and shapes to accommodate pretty much
> any type of coverslip. Some of them have an adhesive side that sticks on
> the slide, then you just need to fill the well (a few 10s-100s of µL will
> do) and gently press the coverslip, then aspirate the excess medium to seal
> the chamber
>
> It is easy to remove the coverslip for further processing, and the
> slide/chamber assembly is reusable. If you worry about the coverslip moving
> during imaging, we have been doing single particle tracking and
> super-resolution microscopy using these chambers without any noticeable
> problems compared to dental cement.
>
> Check here:
> http://www.emsdiasum.com/microscopy/products/preparation/hybridization.aspx
>
> For our 18mm round coverslips we use the one-side adhesive, 13mm wide,
> 0.6mm deep ref. 70-334-A:
>
> http://www.emsdiasum.com/microscopy/products/preparation/hybridization.aspx#70334
>
> Hope this helps,
>
> Christophe
>
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>
>
>
>
> 2014-02-27 16:52 GMT+01:00 Antonio J Pereira <[hidden email]>:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> >
> > Hi all,
> >
> > For some purposes in live cell imaging we assemble a chamber with a
> > coverslip on top of a slide.
> > We use two halves of another coverslip to act as spacers.
> >
> > The  issue is the sealing. We've been using VALAP (a kind of wax) that we
> >  melt and then use to seal all the way through the periphery of the
> >  coverslip.
> >
> > It works, but it would be more convenient to have something that does not
> > have to be heated, and that would harden just by  contact with air.
> > A bit like the nail varnish used for fixed material ... but here we need
> > something which is biologically inert.
> >
> > Any suggestions? Thanks a lot.
> >
> > Antonio
> >
> >
> >
> > António Pereira
> > Chromosome Instability and Dynamics Group, Maiato lab
> >
> > IBMC - Institute for Molecular and Cell Biology
> > Porto- Portugal
> >
> >
> > António Pereira
> > IBMC - Instituto de Biologia Molecular e Celular
> > Grupo Instabilidade e Dinâmica Cromossómica
> >
> > Rua do Campo Alegre, 823
> > 4150-180 Porto
> > Portugal
> >
> > [phone] (ext# 1620)
> > [hidden email]
> >
>

[Kilgore, Jason A.]

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

One thought occurred to me on this thread: how much coverslip sealing is done on live cell preps vs. fixed-cell preps in your labs?

When I've done coverslip sealing (using melted household wax, mainly, though occasionally clear nail polish), it's been on live preps for immediate imaging, but also for non-curing mounting media on fixed cell preps for archiving for a day or so.  Though when I have fixed cell preps and wish to archive, I've typically used a curing mountant.

Thoughts?

Jason

Jason A. Kilgore
Molecular Probes Tech Support
Thermo Fisher Scientific

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Littlejohn, George
Sent: Thursday, February 27, 2014 9:08 AM
To: [hidden email]
Subject: Re: Biological inert coverslip sealing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Antonio,

I can recommend Carolina Observation Gel (polydimethylsiloxane, and supplied by Blades Biological in the UK).

All the best,
George

******************************
Dr. George Littlejohn
Halpin Laboratory,
School of Biosciences,
University of Exeter,
Geoffrey Pope Building,
Stocker Road,
EX4 4QD, UK

Telephone: [phone]
E-mail:  [hidden email]<mailto:[hidden email]>

http://projects.exeter.ac.uk/ein/
https://twitter.com/geolittlejohn


On 27 Feb 2014, at 16:05, "Antonio J Pereira" <[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
*****


Hi all,

For some purposes in live cell imaging we assemble a chamber with a coverslip on top of a slide.
We use two halves of another coverslip to act as spacers.

The  issue is the sealing. We've been using VALAP (a kind of wax) that we  melt and then use to seal all the way through the periphery of the  coverslip.

It works, but it would be more convenient to have something that does not have to be heated, and that would harden just by  contact with air.
A bit like the nail varnish used for fixed material ... but here we need something which is biologically inert.

Any suggestions? Thanks a lot.

Antonio



António Pereira
Chromosome Instability and Dynamics Group, Maiato lab

IBMC - Institute for Molecular and Cell Biology
Porto- Portugal


António Pereira
IBMC - Instituto de Biologia Molecular e Celular
Grupo Instabilidade e Dinâmica Cromossómica

Rua do Campo Alegre, 823
4150-180 Porto
Portugal

[phone] (ext# 1620)
[hidden email]<mailto:[hidden email]>