Coverslip on coverslip - can cells inbetween be imaged?

[Seb Stephens]

We have the problem of our cells differentiating well on specific coverslips,
and not those of mattek dishes. In the meantime of trying to identify other
dishes, we continue to use coverslips.

We image just with a dipping lens, but would like some more resolution.

Is it possible to mount the "coverslip with cells" upsidedown onto a new
mattek dish and then image it using an inverted microscope with a water
immersion lens?

Is there another way around this?

Regards

[Roman Veith]

Hi Waldo,

we use custom made coverslip holder built in our toolshop. This works
very well for us. Cells grow on coverslips and whenever needed single
coverslips with cells are collected from the cell culture. The coverslip
holder is simple a metal device with an adapter for coverslips in the
middle. Imagine the metal device as a metal object slide with a hole in
the middle, designed in a way, that the coverslip exactly fits into it,
without passing through. E.g. for a 18x18 coverslip the hole has
dimensions of ~16x16. Another piece of metal with a ~16 circular whole
in it and a rubber seal is placed on top. Immidiately after transfer of
the coverslips new buffer is added. The rubber seal of the upper device
assures that everything is leak-proof. Maybe after this procedure some
recovery time for the cells is appropriate.
Cells can then be imaged even with an oil immersion objective - if
refractive index mismatch is no problem...

Regards,
Roman


Waldo Schmidt schrieb:

> We have the problem of our cells differentiating well on specific coverslips,
> and not those of mattek dishes. In the meantime of trying to identify other
> dishes, we continue to use coverslips.
>
> We image just with a dipping lens, but would like some more resolution.
>
> Is it possible to mount the "coverslip with cells" upsidedown onto a new
> mattek dish and then image it using an inverted microscope with a water
> immersion lens?
>
> Is there another way around this?
>
> Regards
>  

--
--
Roman Veith
Dipl.-Biol.

Institut für physikalische und
theoretische Chemie
AG Biophysikalische Chemie
Universität Bonn
Wegelerstraße 12
53115 Bonn

Tel-Nr.: 0228-733089
Fax-Nr.: 0228-739424

http://www.thch.uni-bonn.de/pc/kubitscheck/

[Pascal Lorentz-2]

Hi Waldo,

I think what Roman describes is also sold by a company in Switzerland.
They are called Life Imaging Services:
http://www.lis.ch/

No commercial interest, just a satisfied customer.

Best regards

Pascal


Roman Veith schrieb:

> Hi Waldo,
>
> we use custom made coverslip holder built in our toolshop. This works
> very well for us. Cells grow on coverslips and whenever needed single
> coverslips with cells are collected from the cell culture. The
> coverslip holder is simple a metal device with an adapter for
> coverslips in the middle. Imagine the metal device as a metal object
> slide with a hole in the middle, designed in a way, that the coverslip
> exactly fits into it, without passing through. E.g. for a 18x18
> coverslip the hole has dimensions of ~16x16. Another piece of metal
> with a ~16 circular whole in it and a rubber seal is placed on top.
> Immidiately after transfer of the coverslips new buffer is added. The
> rubber seal of the upper device assures that everything is leak-proof.
> Maybe after this procedure some recovery time for the cells is
> appropriate.
> Cells can then be imaged even with an oil immersion objective - if
> refractive index mismatch is no problem...
>
> Regards,
> Roman
>
>
> Waldo Schmidt schrieb:
>> We have the problem of our cells differentiating well on specific
>> coverslips, and not those of mattek dishes. In the meantime of trying
>> to identify other dishes, we continue to use coverslips.
>>
>> We image just with a dipping lens, but would like some more resolution.
>>
>> Is it possible to mount the "coverslip with cells" upsidedown onto a
>> new mattek dish and then image it using an inverted microscope with a
>> water immersion lens?
>> Is there another way around this?
>> Regards
>>  
>
>

Pascal Lorentz
Department of Biomedicine
University of Basel
Mattenstrasse 28
4058 Basel
Switzerland

[Cameron Nowell]

Hi Waldo,
 
Your other choice is to get a Sykes-Moore chanmber made by Belco. The are a simple chamber with a rubber seal and a screw on top that holds it all together. We use them daily and they work great. You can grow your cells on the coverslips then just mount them in the chamber and image happily. The only problem i have had with them is that you cannot usually image to whole coverslip. When mounted the cover slip is recessed a little bit which means large objectives can only focus on the middle area of the cover slip.
 
A big thanks to Steve Cody for buying these in for the Centre. Also I have no comercial intrsts with Belco.
 
 
Cheers
 
Cam
 
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

________________________________

From: Confocal Microscopy List on behalf of Waldo Schmidt
Sent: Wed 26/08/2009 11:31 PM
To: [hidden email]
Subject: Coverslip on coverslip - can cells inbetween be imaged?



We have the problem of our cells differentiating well on specific coverslips,
and not those of mattek dishes. In the meantime of trying to identify other
dishes, we continue to use coverslips.

We image just with a dipping lens, but would like some more resolution.

Is it possible to mount the "coverslip with cells" upsidedown onto a new
mattek dish and then image it using an inverted microscope with a water
immersion lens?

Is there another way around this?

Regards

[Jacqueline Ross]

Hi Waldo,

I know others have already replied with some suggestions but I have one
more for you to consider. I recently bought two chambers from Quorum
Technologies, called Chamlide chambers. They are designed to take
various sizes of coverslip ( I bought the 25mm one) and are magnetic. I
have only just bought them having seen them at the Vancouver LiveCell
course and haven't yet tried them but they seem like they will work
really well.

No commercial interest!

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Waldo Schmidt
Sent: Thursday, 27 August 2009 1:31 a.m.
To: [hidden email]
Subject: Coverslip on coverslip - can cells inbetween be imaged?

We have the problem of our cells differentiating well on specific
coverslips,
and not those of mattek dishes. In the meantime of trying to identify
other
dishes, we continue to use coverslips.

We image just with a dipping lens, but would like some more resolution.

Is it possible to mount the "coverslip with cells" upsidedown onto a new

mattek dish and then image it using an inverted microscope with a water
immersion lens?

Is there another way around this?

Regards

[Alberto Diaspro]

Dear friends,
in my department at the Italian Institute of Technology there are 20  
PhD positions open. A specific one is reported here.
The complete call can be found at www.iit.it.
All the best
Alby

----- Diaspro Lab call ----
Theme 3.1: Optical Nanoscopy and advanced high-resolution microscopy.
Tutor: Alberto Diaspro
A recognized advantage of optical microscopy lies in the fact that  
allows non-invasive three-dimensional (3D)
imaging of live cells at the submicron scale with high specificity.  
The advent of the visible fluorescent proteins
and of a myriad of fluorescent tags pushed fluorescence microscopy to  
become the most popular imaging
tool in cell biology. The confocal and multiphoton versions of  
fluorescence microscopy reinforce this
condition. However, like any other standard imaging system relying on  
focused light, all these microscopes
are limited in spatial resolution because the smallest possible spot  
size is imposed by diffraction. Several
approaches aimed at overcoming the diffraction limit. Stimulated  
Emission Depletion (STED) microscopy is
one of the most promising Optical Nanoscopy approaches allowing an  
ultimate resolution of 7.6 nm in the
optical regime. In STED microscopy, fluorescence emanating from the  
periphery of the focused excitation
beam is suppressed by a second properly shaped beam that depletes the  
excited state population through
stimulated emission. This effectively narrows fluorescent molecule  
signature, the point spread function (PSF)
of the microscope, to permit superresolved images to be acquired. The  
Optical Nanoscopy theme is related
to the development of an original STED architecture based in white  
light laser illumination and to its
characterization for key applications to tracking of molecular events  
in living cells towards the study of
degenerative processes like neuro-diseases, tumor progression and  
nanoparticle toxic effects on biological
systems. Variations on the theme will include Fluorescence Photo-
Activation Light Microscopy (FPALM) and
Single Plane Illumination Microscopy (SPIM) approaches.
For further details concerning the research project, please contact: [hidden email]

------


----------------------------------------
Alberto Diaspro
Head, Nanophysics Unit
Senior Scientist
The Italian Institute of Technology -IIT
Via Morego, 30
16163 - Genova (Italy)
phone: [phone]
mobile: [phone]
fax:   [phone]
http://www.iit.it
[hidden email]

Professor of Applied Physics
Department of Physics
University of Genova
Via Dodecaneso, 33
16146 Genova - Italy
tel.  [phone]
fax. [phone]
http://www.lambs.it
[hidden email]
-------------------------------------------------------

[Seb Stephens]

Hi Jacqui,
 
waldo = me!!!!!!!!
 
I have just ordered these chamlide chambers too. You know theyre on special now? Like 50% off....
 
Speak soon

 

 

Sébastien Stephens

Harvard University School of Dental Medicine
188 Longwood Ave REB 313
Boston, MA 02115
Ph: 617-4327328
Fax: [phone]
[hidden email]

 

> Date: Thu, 27 Aug 2009 09:49:57 +1200
> From: [hidden email]
> Subject: Re: Coverslip on coverslip - can cells inbetween be imaged?
> To: [hidden email]
>
> Hi Waldo,
>
> I know others have already replied with some suggestions but I have one
> more for you to consider. I recently bought two chambers from Quorum
> Technologies, called Chamlide chambers. They are designed to take
> various sizes of coverslip ( I bought the 25mm one) and are magnetic. I
> have only just bought them having seen them at the Vancouver LiveCell
> course and haven't yet tried them but they seem like they will work
> really well.
>
> No commercial interest!
>
> Kind regards,
>
> Jacqui
>
> Jacqueline Ross
>
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland, NEW ZEALAND
>
> Tel: [phone] Ext 87438
> Fax: [phone]
>
> http://www.fmhs.auckland.ac.nz/sms/biru/
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Waldo Schmidt
> Sent: Thursday, 27 August 2009 1:31 a.m.
> To: [hidden email]
> Subject: Coverslip on coverslip - can cells inbetween be imaged?
>
> We have the problem of our cells differentiating well on specific
> coverslips,
> and not those of mattek dishes. In the meantime of trying to identify
> other
> dishes, we continue to use coverslips.
>
> We image just with a dipping lens, but would like some more resolution.
>
> Is it possible to mount the "coverslip with cells" upsidedown onto a new
>
> mattek dish and then image it using an inverted microscope with a water
> immersion lens?
>
> Is there another way around this?
>
> Regards

---

Partagez vos souvenirs sur le Web avec les personnes de votre choix les personnes de votre choix.

[Seb Stephens]

I tried today mounting a "coverslip+cells" upsidedown on another coverslip, all covered in media...
 
Cells died in a few hours....

---

Partagez vos souvenirs sur le Web avec les personnes de votre choix les personnes de votre choix.

[Mayandi Sivaguru]

Try, ibidi dishes ibidi.com no commercial interest.
Shiv

> I tried today mounting a "coverslip+cells" upsidedown on another
> coverslip, all covered in media...
>
>
>
> Cells died in a few hours....
>
> _________________________________________________________________
> Partagez vos souvenirs sur le Web avec les personnes de votre choix.
> http://www.microsoft.com/northafrica/windows/windowslive/products/photos-share.aspx?tab=1

[Keith Morris]

I'd also recommend the trying the plastic cover-slip thickness membrane
slides and wells from Ibidi:


Ibidi μ-well MultiWell, micro-channel and Flask 3x1" slides [new product]
for slide cell culture and hi-res microscopy imaging. Go to Ibidis web site.
Wells and channels with a 150μm membrane cover-slip on a standard slide
http://www.ibidi.de/products/p_disposables.html


Griener also make utra-thin Lumox membrane [as well as glass or plastic]
wells and slides for inverted microscope viewing:
Greiner bio-one MultiWell and SlideFlask slides [new product] for slide cell
culture and microscopy imaging. Download Greiner Culture Slide Brochure. 1
to 8 wells on a Lumox membrane, cover-slip or glass slide
http://www.well.ox.ac.uk/cytogenetics/downloads/Griener MultiWell & Flask
Slides.pdf


You can of course buy the Mattek dishes as uncoated, poly-d-lysine coated,
or collagen coated, plus they offer two cover-slip surfaces [standard glass
and plastic CS1 - the latter's unsuitable for oil immersion though] that
might help.
http://www.glass-bottom-dishes.com/faq.html#typedish
http://www.glass-bottom-dishes.com/coverslips.html

Our workshop back at the Institute of Ophthalmology, UCL, did try making our
own 'Mattek' style dishes with coverslips of our choice, but it was hard
cutting out the circle at the base of the Petri dish and they gave up. I did
consider laser cutting out-sourcing to make the hole [that we used very
successfully for cutting our plastic CR-39 autoradiography 3x1" slides years
ago] and applying the cover-slip of choice, but it was easier just buying in
the Mattek dishes as they generally worked OK for our cell biologists.

Most suppliers offer free samples to try out.

Regards

Keith

For more web-links see
http://www.well.ox.ac.uk/external-website-links

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of [hidden email]
Sent: 27 August 2009 03:59
To: [hidden email]
Subject: Re: Coverslip on coverslip - can cells inbetween be imaged?

Try, ibidi dishes ibidi.com no commercial interest.
Shiv

> I tried today mounting a "coverslip+cells" upsidedown on another
> coverslip, all covered in media...
>
>
>
> Cells died in a few hours....
>
> _________________________________________________________________
> Partagez vos souvenirs sur le Web avec les personnes de votre choix.
>

http://www.microsoft.com/northafrica/windows/windowslive/products/photos-sha
re.aspx?tab=1

[laiding]

maybe use a couple of coverslip as spacer, so the cells won't be under pressure.

 

good luck.

Lai

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of Seb Stephens [[hidden email]]
Sent: Wednesday, August 26, 2009 9:56 PM
To: [hidden email]
Subject:

I tried today mounting a "coverslip+cells" upsidedown on another coverslip, all covered in media...
 
Cells died in a few hours....

---

Partagez vos souvenirs sur le Web avec les personnes de votre choix les personnes de votre choix.

[Ignatius, Mike-2]

Hi Waldo,

One more suggestion.

We have been selling the AttoFluor chamber (A7816) for over a decade for this application as well.  Your coverslip (25mm round) and our holder.  Very simple, autoclavable design.

Regards,

Mike Ignatius,

Molecular Probes/Life Technologies.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Wednesday, August 26, 2009 2:18 PM
To: [hidden email]
Subject: Re: Coverslip on coverslip - can cells inbetween be imaged?

Hi Waldo,
 
Your other choice is to get a Sykes-Moore chanmber made by Belco. The are a simple chamber with a rubber seal and a screw on top that holds it all together. We use them daily and they work great. You can grow your cells on the coverslips then just mount them in the chamber and image happily. The only problem i have had with them is that you cannot usually image to whole coverslip. When mounted the cover slip is recessed a little bit which means large objectives can only focus on the middle area of the cover slip.
 
A big thanks to Steve Cody for buying these in for the Centre. Also I have no comercial intrsts with Belco.
 
 
Cheers
 
Cam
 
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

________________________________

From: Confocal Microscopy List on behalf of Waldo Schmidt
Sent: Wed 26/08/2009 11:31 PM
To: [hidden email]
Subject: Coverslip on coverslip - can cells inbetween be imaged?



We have the problem of our cells differentiating well on specific coverslips,
and not those of mattek dishes. In the meantime of trying to identify other
dishes, we continue to use coverslips.

We image just with a dipping lens, but would like some more resolution.

Is it possible to mount the "coverslip with cells" upsidedown onto a new
mattek dish and then image it using an inverted microscope with a water
immersion lens?

Is there another way around this?

Regards