Live cell FRET - Glass or Plastic?

[Pablo German]

Hi Everyone,

 

I'm in the process of doing FRET experiments using the pair CFP-YFP in live cells and I was wondering whether glass-bottomed dishes are better than plastic dishes for confocal imaging. HEK cells don't seem to want to attach to glass-bottomed dishes even when they are coated with poly-lysine, so using plastic dishes would make things easier.

 

Thanks in advance,

Pablo

--
Pablo German

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]

[Boswell, Carl A - (cboswell)]

Could there be a polarizing issue, given that light from the laser may be polarized and the polarizing effects inherent in manufactured plastic plates?

carl

 

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Wednesday, January 21, 2009 5:02 PM

Subject: Live cell FRET - Glass or Plastic?

Hi Everyone,

 

I'm in the process of doing FRET experiments using the pair CFP-YFP in live cells and I was wondering whether glass-bottomed dishes are better than plastic dishes for confocal imaging. HEK cells don't seem to want to attach to glass-bottomed dishes even when they are coated with poly-lysine, so using plastic dishes would make things easier.

 

Thanks in advance,

Pablo

--
Pablo German

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]

[vaishali kailaje]

Hi,

Glass bottom plates are any time preferable than plastic bottom for live cell imaging at higher magnification as you will not face any problems while focusing as well as there would be no loss in intensity.

Regarding growing cells , we have experienced that cells adhere very well when serum containing medium is used rather than in serum free medium.Probably you could try the same.

Best Luck.

Vaishali



--- On Thu, 1/22/09, Pablo German <[hidden email]> wrote:

> From: Pablo German <[hidden email]>
> Subject: Live cell FRET - Glass or Plastic?
> To: [hidden email]
> Date: Thursday, January 22, 2009, 5:32 AM
> Hi Everyone,
>
> I'm in the process of doing FRET experiments using the
> pair CFP-YFP in live
> cells and I was wondering whether glass-bottomed dishes are
> better than
> plastic dishes for confocal imaging. HEK cells don't
> seem to want to attach
> to glass-bottomed dishes even when they are coated with
> poly-lysine, so
> using plastic dishes would make things easier.
>
> Thanks in advance,
> Pablo
>
> --
> Pablo German
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: [phone]
> Mobile: [phone]

     

[Csúcs Gábor]

Hallo Pablo,

You may try the Ibidi chambers/slides although I have no idea whether
the are represented in the southern hemisphere. They have a thin plastic
bottom but concerning imaging you can reach nearly the same quality as
with glass.

Cheers    Gabor

No commercial interest.

> Hi Everyone,
>  
> I'm in the process of doing FRET experiments using the pair CFP-YFP in
> live cells and I was wondering whether glass-bottomed dishes are
> better than plastic dishes for confocal imaging. HEK cells don't seem
> to want to attach to glass-bottomed dishes even when they are coated
> with poly-lysine, so using plastic dishes would make things easier.
>  
> Thanks in advance,
> Pablo
>
> --
> Pablo German
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: [phone]
> Mobile: [phone]

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: [phone]
Fax: [phone]
e-mail: [hidden email]

[Periasamy, Ammasi (ap3t)]

If you are using laser as an excitation source plastic is not a good idea, you get high light scattering. If you are using arc lamp light source its ok to use good quality petri dish. It is important to understand that you are trying to collect FRET signal, as you know thats very weak signal compared to its fluorescence signal. you may see some artifacts of increase in signal in the acceptor channel when you excite with donor excitation wavelength.
Hope this helps

Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI)
Professor of Biology and Biomedical Engineering
Biology, Gilmer Hall (064), McCormick Rd
University of Virginia
Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http//:www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 3-7, 2009
http://www.kcci.virginia.edu/workshop/workshop2009/index.php
 *************************
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Gabor Csucs [[hidden email]]
Sent: Thursday, January 22, 2009 3:35 AM
To: [hidden email]
Subject: Re: Live cell FRET - Glass or Plastic?

Hallo Pablo,

You may try the Ibidi chambers/slides although I have no idea whether
the are represented in the southern hemisphere. They have a thin plastic
bottom but concerning imaging you can reach nearly the same quality as
with glass.

Cheers    Gabor

No commercial interest.

> Hi Everyone,
>
> I'm in the process of doing FRET experiments using the pair CFP-YFP in
> live cells and I was wondering whether glass-bottomed dishes are
> better than plastic dishes for confocal imaging. HEK cells don't seem
> to want to attach to glass-bottomed dishes even when they are coated
> with poly-lysine, so using plastic dishes would make things easier.
>
> Thanks in advance,
> Pablo
>
> --
> Pablo German
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: [phone]
> Mobile: [phone]

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: [phone]
Fax: [phone]
e-mail: [hidden email]

[Craig Brideau]

As everyone else is saying; glass has a much better optical quality than most plastics.  It also tends to be flatter.

Craig

On Thu, Jan 22, 2009 at 7:49 AM, Periasamy, Ammasi (ap3t) <[hidden email]> wrote:

If you are using laser as an excitation source plastic is not a good idea, you get high light scattering. If you are using arc lamp light source its ok to use good quality petri dish. It is important to understand that you are trying to collect FRET signal, as you know thats very weak signal compared to its fluorescence signal. you may see some artifacts of increase in signal in the acceptor channel when you excite with donor excitation wavelength.
Hope this helps

Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI)
Professor of Biology and Biomedical Engineering
Biology, Gilmer Hall (064), McCormick Rd
University of Virginia
Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; [hidden email]
http//:www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 3-7, 2009
http://www.kcci.virginia.edu/workshop/workshop2009/index.php
 *************************
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Gabor Csucs [[hidden email]]
Sent: Thursday, January 22, 2009 3:35 AM

To: [hidden email]

Subject: Re: Live cell FRET - Glass or Plastic?

Hallo Pablo,

You may try the Ibidi chambers/slides although I have no idea whether
the are represented in the southern hemisphere. They have a thin plastic
bottom but concerning imaging you can reach nearly the same quality as
with glass.

Cheers    Gabor

No commercial interest.
> Hi Everyone,
>
> I'm in the process of doing FRET experiments using the pair CFP-YFP in
> live cells and I was wondering whether glass-bottomed dishes are
> better than plastic dishes for confocal imaging. HEK cells don't seem
> to want to attach to glass-bottomed dishes even when they are coated
> with poly-lysine, so using plastic dishes would make things easier.
>
> Thanks in advance,
> Pablo
>
> --
> Pablo German
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: [phone]
> Mobile: [phone]


--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: [phone]
Fax: [phone]
e-mail: [hidden email]

[Megan Nicholson]

Hi Pablo,

ibidi Slides would work great as they have a very thin plastic bottom with the
optical qualitiy of a 1.5 glass coverslip.  You can get up to three free samples
to try and there is a distributer in Austrailia:
DKSH Australia Pty. Ltd.
14-17 Dansu Court
Hallam Vic 3803
Phone: [phone]
Fax: [phone]
Email: [hidden email]
www.dksh.com/australia/ lifesciences

You can either email them or go to the website www.ibidi.com and request
them.

Good luck!

Megan