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Mounting media for FRET experiments

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Romin, Yevgeniy/Sloan Kettering Institute

Mounting media for FRET experiments

Dear list

During a recent FRET experiment a question came up about what mounting media should be used on fixed samples during FRET experiments and whether any one media is better than other for this particular type of experiment. From what’s published it seems that people use all kinds of different media. Does anybody here have any idea about any specific media that would be best/give least interference with FRET results?

Thanks a lot to all of you in advance,

---------------------------------------------------

Yevgeniy Romin

Digital Microscopist

Memorial Sloan-Kettering Cancer Center

Molecular Cytology Core Facility

1275 York Ave. Box 333

New York, NY 10065

Tel.646-888-2186

Fax. 646-422-0640

---------------------------------------------------

 
     =====================================================================
     
     Please note that this e-mail and any files transmitted with it may be 
     privileged, confidential, and protected from disclosure under 
     applicable law. If the reader of this message is not the intended 
     recipient, or an employee or agent responsible for delivering this 
     message to the intended recipient, you are hereby notified that any 
     reading, dissemination, distribution, copying, or other use of this 
     communication or any of its attachments is strictly prohibited.  If 
     you have received this communication in error, please notify the 
     sender immediately by replying to this message and deleting this 
     message, any attachments, and all copies and backups from your 
     computer.

John Oreopoulos

Re: Mounting media for FRET experiments

I have not used fixed specimens for FRET imaging (I have used the sensitized emission method on living cells), but I came across this paper a while ago which might be of interest to you:

Rodighiero, S., et al., Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching. Cellular Physiology and Biochemistry, 2008. 21(5-6): p. 489-498.

John Oreopoulos

On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:

> Dear list
>
> During a recent FRET experiment a question came up about what mounting media should be used on fixed samples during FRET experiments and whether any one media is better than other for this particular type of experiment. From what’s published it seems that people use all kinds of different media. Does anybody here have any idea about any specific media that would be best/give least interference with FRET results?
>
> Thanks a lot to all of you in advance,
>
> ---------------------------------------------------
> Yevgeniy Romin
>
> Digital Microscopist
> Memorial Sloan-Kettering Cancer Center
> Molecular Cytology Core Facility
> 1275 York Ave. Box 333
> New York, NY 10065
> Tel.646-888-2186
> Fax. 646-422-0640
> ---------------------------------------------------
>
>
>
> =====================================================================
>
> Please note that this e-mail and any files transmitted with it may be
> privileged, confidential, and protected from disclosure under
> applicable law. If the reader of this message is not the intended
> recipient, or an employee or agent responsible for delivering this
> message to the intended recipient, you are hereby notified that any
> reading, dissemination, distribution, copying, or other use of this
> communication or any of its attachments is strictly prohibited. If
> you have received this communication in error, please notify the
> sender immediately by replying to this message and deleting this
> message, any attachments, and all copies and backups from your
> computer.
>
>

Romin, Yevgeniy/Sloan Kettering Institute

Re: Mounting media for FRET experiments

Thank you very much John, I found the paper you recommended and it is already proving useful. A question for you then - which media were you using for the live cells? What I mean is whether it was something like PBS or some specific media?

---------------------------------------------------
Yevgeniy Romin

Digital Microscopist
Memorial Sloan-Kettering Cancer Center
Molecular Cytology Core Facility
1275 York Ave. Box 333
New York, NY 10065
Tel.646-888-2186
Fax. 646-422-0640
---------------------------------------------------

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Friday, April 16, 2010 11:30 AM
To: [hidden email]
Subject: Re: Mounting media for FRET experiments

I have not used fixed specimens for FRET imaging (I have used the sensitized emission method on living cells), but I came across this paper a while ago which might be of interest to you:

Rodighiero, S., et al., Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching. Cellular Physiology and Biochemistry, 2008. 21(5-6): p. 489-498.

John Oreopoulos

On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:

> Dear list
>
> During a recent FRET experiment a question came up about what mounting media should be used on fixed samples during FRET experiments and whether any one media is better than other for this particular type of experiment. From what's published it seems that people use all kinds of different media. Does anybody here have any idea about any specific media that would be best/give least interference with FRET results?
>
> Thanks a lot to all of you in advance,
>
> ---------------------------------------------------
> Yevgeniy Romin
>
> Digital Microscopist
> Memorial Sloan-Kettering Cancer Center
> Molecular Cytology Core Facility
> 1275 York Ave. Box 333
> New York, NY 10065
> Tel.646-888-2186
> Fax. 646-422-0640
> ---------------------------------------------------
>
>
>
> =====================================================================
>
> Please note that this e-mail and any files transmitted with it may be
> privileged, confidential, and protected from disclosure under
> applicable law. If the reader of this message is not the intended
> recipient, or an employee or agent responsible for delivering this
> message to the intended recipient, you are hereby notified that any
> reading, dissemination, distribution, copying, or other use of this
> communication or any of its attachments is strictly prohibited. If
> you have received this communication in error, please notify the
> sender immediately by replying to this message and deleting this
> message, any attachments, and all copies and backups from your
> computer.
>
>

John Oreopoulos

Re: Mounting media for FRET experiments

Just regular PBS solution. I have never tried a rigorous comparison of different media.

John

On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:

> Thank you very much John, I found the paper you recommended and it is already proving useful. A question for you then - which media were you using for the live cells? What I mean is whether it was something like PBS or some specific media?
>
> ---------------------------------------------------
> Yevgeniy Romin
>
> Digital Microscopist
> Memorial Sloan-Kettering Cancer Center
> Molecular Cytology Core Facility
> 1275 York Ave. Box 333
> New York, NY 10065
> Tel.646-888-2186
> Fax. 646-422-0640
> ---------------------------------------------------
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
> Sent: Friday, April 16, 2010 11:30 AM
> To: [hidden email]
> Subject: Re: Mounting media for FRET experiments
>
> I have not used fixed specimens for FRET imaging (I have used the sensitized emission method on living cells), but I came across this paper a while ago which might be of interest to you:
>
> Rodighiero, S., et al., Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching. Cellular Physiology and Biochemistry, 2008. 21(5-6): p. 489-498.
>
> John Oreopoulos
>
>
> On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
>
>> Dear list
>>
>> During a recent FRET experiment a question came up about what mounting media should be used on fixed samples during FRET experiments and whether any one media is better than other for this particular type of experiment. From what's published it seems that people use all kinds of different media. Does anybody here have any idea about any specific media that would be best/give least interference with FRET results?
>>
>> Thanks a lot to all of you in advance,
>>
>> ---------------------------------------------------
>> Yevgeniy Romin
>>
>> Digital Microscopist
>> Memorial Sloan-Kettering Cancer Center
>> Molecular Cytology Core Facility
>> 1275 York Ave. Box 333
>> New York, NY 10065
>> Tel.646-888-2186
>> Fax. 646-422-0640
>> ---------------------------------------------------
>>
>>
>>
>> =====================================================================
>>
>> Please note that this e-mail and any files transmitted with it may be
>> privileged, confidential, and protected from disclosure under
>> applicable law. If the reader of this message is not the intended
>> recipient, or an employee or agent responsible for delivering this
>> message to the intended recipient, you are hereby notified that any
>> reading, dissemination, distribution, copying, or other use of this
>> communication or any of its attachments is strictly prohibited. If
>> you have received this communication in error, please notify the
>> sender immediately by replying to this message and deleting this
>> message, any attachments, and all copies and backups from your
>> computer.
>>
>>

Vitaly Boyko

Re: Mounting media for FRET experiments

Dear John,

Dear Yevgeniy,

Yes, PBS or specialized low serum (1-2%) media is broadly used for live cell imaging under TIR illumination.

I would like to bring your attention to two papers which are relevant to single molecule fluorscence and/or low light live cell imaging - I sent them separately to your e-mail addressess as the LISTSERVER rejected my message due to the attachments. I can also send the papers to anyone by request.

1. The Use of Protocatechuate Dioxygenase for Maintaining Anaerobic Conditions in Biochemical Experiments

Pravin V. Patil and David P. Ballou

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606

Analytical Biochemistry 286, 187–192 (2000)

2. An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments

Colin Echeverrı´a Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§

Biophysical Journal Volume 94 March 2008 1826–1835

If you have any questions, please contact me offline or phone.

Also, the accuracy of the FRET eff. measurements could vary or be affected by the mounting media used - live cell FRET seems to be the best approach.

Have a nice weekend,

Vitaly

301-515-7833

From: John Oreopoulos <[hidden email]>
To: [hidden email]
Sent: Fri, April 16, 2010 12:01:32 PM
Subject: Re: Mounting media for FRET experiments

Just regular PBS solution. I have never tried a rigorous comparison of different media.

John

On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:

Daniel Gitler

Re: Mounting media for FRET experiments

Dear John and Yevgeniy,

My experience when measuring FRET from the same samples before and after fixation is that fixation itself will very much change the FRET efficiency value that you measure (Cerulean and Venus in HEK cells, PBS for both). Efficiency in the fixed samples was significantly lower. This should not come as any big surprise, since the cellular environment around the fluorophores will be very different.
The possible reasons for the changes in FRET efficiency are very many...
I also do most of my FRET imaging in live samples.

Daniel

----- Original Message -----
From: Vitaly Boyko <[hidden email]>
Date: Friday, April 16, 2010 19:36
Subject: Re: Mounting media for FRET experiments
To: [hidden email]

> Dear John,
> Dear Yevgeniy,
>
> Yes, PBS or specialized low serum (1-2%) media is broadly used
> for live cell imaging under TIR illumination.
>
> I would like to bring your attention to two papers which are
> relevant to single molecule fluorscence and/or low light live
> cell imaging - I sent them separately to your e-mail addressess
> as the LISTSERVER rejected my message due to the attachments. I
> can also send the papers to anyone by request.
>
> 1. The Use of Protocatechuate Dioxygenase for Maintaining
> Anaerobic Conditions in Biochemical ExperimentsPravin V. Patil
> and David P. BallouDepartment of Biological Chemistry,
> University of Michigan, Ann Arbor, Michigan 48109-0606
> Analytical Biochemistry 286, 187–192 (2000)
> 2. An Oxygen Scavenging System for Improvement of Dye Stability
> in Single-Molecule Fluorescence ExperimentsColin Echeverrı´a
> Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§
> Biophysical Journal Volume 94 March 2008 1826–1835
>
> If you have any questions, please contact me offline or phone.
>
> Also, the accuracy of the FRET eff. measurements could vary or
> be affected by the mounting media used - live cell FRET seems to
> be the best approach.
>
> Have a nice weekend,
>
> Vitaly
> 301-515-7833
>
>
>
>
>
> ________________________________
> From: John Oreopoulos <[hidden email]>
> To: [hidden email]
> Sent: Fri, April 16, 2010 12:01:32 PM
> Subject: Re: Mounting media for FRET experiments
>
> Just regular PBS solution. I have never tried a rigorous
> comparison of different media.
>
> John
>
> On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:
>
> > Thank you very much John, I found the paper you recommended
> and it is already proving useful. A question for you then -
> which media were you using for the live cells? What I mean is
> whether it was something like PBS or some specific media?
> >
> > ---------------------------------------------------
> > Yevgeniy Romin
> >
> > Digital Microscopist
> > Memorial Sloan-Kettering Cancer Center
> > Molecular Cytology Core Facility
> > 1275 York Ave. Box 333
> > New York, NY 10065
> > Tel.646-888-2186
> > Fax. 646-422-0640
> > ---------------------------------------------------
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of John Oreopoulos
> > Sent: Friday, April 16, 2010 11:30 AM
> > To: [hidden email]
> > Subject: Re: Mounting media for FRET experiments
> >
> > I have not used fixed specimens for FRET imaging (I have used
> the sensitized emission method on living cells), but I came
> across this paper a while ago which might be of interest to you:
> >
> > Rodighiero, S., et al., Fixation, mounting and sealing with
> nail polish of cell specimens lead to incorrect FRET
> measurements using acceptor photobleaching. Cellular Physiology
> and Biochemistry, 2008. 21(5-6): p. 489-498.
> >
> > John Oreopoulos
> >
> >
> > On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
> >
> >> Dear list
> >>
> >> During a recent FRET experiment a question came up about what
> mounting media should be used on fixed samples during FRET
> experiments and whether any one media is better than other for
> this particular type of experiment. From what's published it
> seems that people use all kinds of different media. Does
> anybody here have any idea about any specific media that would
> be best/give least interference with FRET results?
> >>
> >> Thanks a lot to all of you in advance,
> >>
> >> ---------------------------------------------------
> >> Yevgeniy Romin
> >>
> >> Digital Microscopist
> >> Memorial Sloan-Kettering Cancer Center
> >> Molecular Cytology Core Facility
> >> 1275 York Ave. Box 333
> >> New York, NY 10065
> >> Tel.646-888-2186
> >> Fax. 646-422-0640
> >> ---------------------------------------------------
> >>
> >>
> >>
> >>
> =====================================================================>>
> >> Please note that this e-mail and any files transmitted
> with it may be
> >> privileged, confidential, and protected from disclosure under
> >> applicable law. If the reader of this message is not the
> intended>> recipient, or an employee or agent responsible for
> delivering this
> >> message to the intended recipient, you are hereby notified
> that any
> >> reading, dissemination, distribution, copying, or other
> use of this
> >> communication or any of its attachments is strictly
> prohibited. If
> >> you have received this communication in error, please
> notify the
> >> sender immediately by replying to this message and
> deleting this
> >> message, any attachments, and all copies and backups from your
> >> computer.
> >>
> >>
>
>
>
>

Daniel Gitler, Ph.D.
Department of Physiology and Neurobiology
Faculty of Health Sciences
Ben Gurion University of the Negev
Beer-Sheva 84105
Israel

Tel: +972-8-6477345
Cell: +972-54-2110100
Fax: + 972-8-6477628
http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/

‎

Romin, Yevgeniy/Sloan Kettering Institute

Re: Mounting media for FRET experiments

Thank you very much to everyone replying both on and off the list. Your advice and the papers that were referred to were very helpful. FRET on live samples would be the ideal choice, unfortunately in this case we are doing FRET on white blood cells, which are very round and refuse to attach and sit still, introducing motion artifacts into the FRET analysis. We are currently working on immobilizing them, but there is a fear of matrigel or a more viscous media introducing FRET measurement artifacts as well. Does anyone have experience with doing FRET on cells that do not attach or immobilizing them in a least invasive way?
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Daniel Gitler [[hidden email]]
Sent: Saturday, April 17, 2010 5:22 AM
To: [hidden email]
Subject: Re: Mounting media for FRET experiments

Dear John and Yevgeniy,

My experience when measuring FRET from the same samples before and after fixation is that fixation itself will very much change the FRET efficiency value that you measure (Cerulean and Venus in HEK cells, PBS for both). Efficiency in the fixed samples was significantly lower. This should not come as any big surprise, since the cellular environment around the fluorophores will be very different.
The possible reasons for the changes in FRET efficiency are very many...
I also do most of my FRET imaging in live samples.

Daniel

----- Original Message -----
From: Vitaly Boyko <[hidden email]>
Date: Friday, April 16, 2010 19:36
Subject: Re: Mounting media for FRET experiments
To: [hidden email]

Cameron Nowell

Re: Mounting media for FRET experiments

Dear Yevgeniy,

While i haven't had any experience imobilising cells for FRET we have had great success in the past imobolising cells for ICS. We had a cell line that expressed the receptor of interest but was round and didn't attache to anything. We just put them in some 1% low melt temp agarose (made up in PBS) and everything worked really well. Now we were only looking at the bleching of one receptor, but any movement in the system would have been noticed in the final analysis and there was none. I cant see a low percentage of agarose interfereing with you FRET measurments.

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: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm

________________________________

From: Confocal Microscopy List on behalf of Yevgeniy Romin
Sent: Sun 18/04/2010 1:49 AM
To: [hidden email]
Subject: Re: Mounting media for FRET experiments

Thank you very much to everyone replying both on and off the list. Your advice and the papers that were referred to were very helpful. FRET on live samples would be the ideal choice, unfortunately in this case we are doing FRET on white blood cells, which are very round and refuse to attach and sit still, introducing motion artifacts into the FRET analysis. We are currently working on immobilizing them, but there is a fear of matrigel or a more viscous media introducing FRET measurement artifacts as well. Does anyone have experience with doing FRET on cells that do not attach or immobilizing them in a least invasive way?
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Daniel Gitler [[hidden email]]
Sent: Saturday, April 17, 2010 5:22 AM
To: [hidden email]
Subject: Re: Mounting media for FRET experiments

Dear John and Yevgeniy,

My experience when measuring FRET from the same samples before and after fixation is that fixation itself will very much change the FRET efficiency value that you measure (Cerulean and Venus in HEK cells, PBS for both). Efficiency in the fixed samples was significantly lower. This should not come as any big surprise, since the cellular environment around the fluorophores will be very different.
The possible reasons for the changes in FRET efficiency are very many...
I also do most of my FRET imaging in live samples.

Daniel

----- Original Message -----
From: Vitaly Boyko <[hidden email]>
Date: Friday, April 16, 2010 19:36
Subject: Re: Mounting media for FRET experiments
To: [hidden email]

> Dear John,
> Dear Yevgeniy,
>
> Yes, PBS or specialized low serum (1-2%) media is broadly used
> for live cell imaging under TIR illumination.
>
> I would like to bring your attention to two papers which are
> relevant to single molecule fluorscence and/or low light live
> cell imaging - I sent them separately to your e-mail addressess
> as the LISTSERVER rejected my message due to the attachments. I
> can also send the papers to anyone by request.
>
> 1. The Use of Protocatechuate Dioxygenase for Maintaining
> Anaerobic Conditions in Biochemical ExperimentsPravin V. Patil
> and David P. BallouDepartment of Biological Chemistry,
> University of Michigan, Ann Arbor, Michigan 48109-0606
> Analytical Biochemistry 286, 187-192 (2000)
> 2. An Oxygen Scavenging System for Improvement of Dye Stability
> in Single-Molecule Fluorescence ExperimentsColin Echeverri´a
> Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§
> Biophysical Journal Volume 94 March 2008 1826-1835
>
> If you have any questions, please contact me offline or phone.
>
> Also, the accuracy of the FRET eff. measurements could vary or
> be affected by the mounting media used - live cell FRET seems to
> be the best approach.
>
> Have a nice weekend,
>
> Vitaly
> 301-515-7833
>
>
>
>
>
> ________________________________
> From: John Oreopoulos <[hidden email]>
> To: [hidden email]
> Sent: Fri, April 16, 2010 12:01:32 PM
> Subject: Re: Mounting media for FRET experiments
>
> Just regular PBS solution. I have never tried a rigorous
> comparison of different media.
>
> John
>
> On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:
>
> > Thank you very much John, I found the paper you recommended
> and it is already proving useful. A question for you then -
> which media were you using for the live cells? What I mean is
> whether it was something like PBS or some specific media?
> >
> > ---------------------------------------------------
> > Yevgeniy Romin
> >
> > Digital Microscopist
> > Memorial Sloan-Kettering Cancer Center
> > Molecular Cytology Core Facility
> > 1275 York Ave. Box 333
> > New York, NY 10065
> > Tel.646-888-2186
> > Fax. 646-422-0640
> > ---------------------------------------------------
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of John Oreopoulos
> > Sent: Friday, April 16, 2010 11:30 AM
> > To: [hidden email]
> > Subject: Re: Mounting media for FRET experiments
> >
> > I have not used fixed specimens for FRET imaging (I have used
> the sensitized emission method on living cells), but I came
> across this paper a while ago which might be of interest to you:
> >
> > Rodighiero, S., et al., Fixation, mounting and sealing with
> nail polish of cell specimens lead to incorrect FRET
> measurements using acceptor photobleaching. Cellular Physiology
> and Biochemistry, 2008. 21(5-6): p. 489-498.
> >
> > John Oreopoulos
> >
> >
> > On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
> >
> >> Dear list
> >>
> >> During a recent FRET experiment a question came up about what
> mounting media should be used on fixed samples during FRET
> experiments and whether any one media is better than other for
> this particular type of experiment. From what's published it
> seems that people use all kinds of different media. Does
> anybody here have any idea about any specific media that would
> be best/give least interference with FRET results?
> >>
> >> Thanks a lot to all of you in advance,
> >>
> >> ---------------------------------------------------
> >> Yevgeniy Romin
> >>
> >> Digital Microscopist
> >> Memorial Sloan-Kettering Cancer Center
> >> Molecular Cytology Core Facility
> >> 1275 York Ave. Box 333
> >> New York, NY 10065
> >> Tel.646-888-2186
> >> Fax. 646-422-0640
> >> ---------------------------------------------------
> >>
> >>
> >>
> >>
> =====================================================================>>
> >> Please note that this e-mail and any files transmitted
> with it may be
> >> privileged, confidential, and protected from disclosure under
> >> applicable law. If the reader of this message is not the
> intended>> recipient, or an employee or agent responsible for
> delivering this
> >> message to the intended recipient, you are hereby notified
> that any
> >> reading, dissemination, distribution, copying, or other
> use of this
> >> communication or any of its attachments is strictly
> prohibited. If
> >> you have received this communication in error, please
> notify the
> >> sender immediately by replying to this message and
> deleting this
> >> message, any attachments, and all copies and backups from your
> >> computer.
> >>
> >>
>
>
>
>

Cameron Nowell

Re: Mounting media for FRET experiments

I should also add that we capture a series first in reflection mode to be able to pick the cells that were against the coverslip to remove and potential artifact of imaging through a chunk of agarose.

Cheers

Cam

________________________________

From: Confocal Microscopy List on behalf of Cameron Nowell
Sent: Sun 18/04/2010 8:47 AM
To: [hidden email]
Subject: Re: Mounting media for FRET experiments

Dear Yevgeniy,

While i haven't had any experience imobilising cells for FRET we have had great success in the past imobolising cells for ICS. We had a cell line that expressed the receptor of interest but was round and didn't attache to anything. We just put them in some 1% low melt temp agarose (made up in PBS) and everything worked really well. Now we were only looking at the bleching of one receptor, but any movement in the system would have been noticed in the final analysis and there was none. I cant see a low percentage of agarose interfereing with you FRET measurments.

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: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm

________________________________

From: Confocal Microscopy List on behalf of Yevgeniy Romin
Sent: Sun 18/04/2010 1:49 AM
To: [hidden email]
Subject: Re: Mounting media for FRET experiments

Thank you very much to everyone replying both on and off the list. Your advice and the papers that were referred to were very helpful. FRET on live samples would be the ideal choice, unfortunately in this case we are doing FRET on white blood cells, which are very round and refuse to attach and sit still, introducing motion artifacts into the FRET analysis. We are currently working on immobilizing them, but there is a fear of matrigel or a more viscous media introducing FRET measurement artifacts as well. Does anyone have experience with doing FRET on cells that do not attach or immobilizing them in a least invasive way?
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Daniel Gitler [[hidden email]]
Sent: Saturday, April 17, 2010 5:22 AM
To: [hidden email]
Subject: Re: Mounting media for FRET experiments

Dear John and Yevgeniy,

My experience when measuring FRET from the same samples before and after fixation is that fixation itself will very much change the FRET efficiency value that you measure (Cerulean and Venus in HEK cells, PBS for both). Efficiency in the fixed samples was significantly lower. This should not come as any big surprise, since the cellular environment around the fluorophores will be very different.
The possible reasons for the changes in FRET efficiency are very many...
I also do most of my FRET imaging in live samples.

Daniel

----- Original Message -----
From: Vitaly Boyko <[hidden email]>
Date: Friday, April 16, 2010 19:36
Subject: Re: Mounting media for FRET experiments
To: [hidden email]

> Dear John,
> Dear Yevgeniy,
>
> Yes, PBS or specialized low serum (1-2%) media is broadly used
> for live cell imaging under TIR illumination.
>
> I would like to bring your attention to two papers which are
> relevant to single molecule fluorscence and/or low light live
> cell imaging - I sent them separately to your e-mail addressess
> as the LISTSERVER rejected my message due to the attachments. I
> can also send the papers to anyone by request.
>
> 1. The Use of Protocatechuate Dioxygenase for Maintaining
> Anaerobic Conditions in Biochemical ExperimentsPravin V. Patil
> and David P. BallouDepartment of Biological Chemistry,
> University of Michigan, Ann Arbor, Michigan 48109-0606
> Analytical Biochemistry 286, 187-192 (2000)
> 2. An Oxygen Scavenging System for Improvement of Dye Stability
> in Single-Molecule Fluorescence ExperimentsColin Echeverri´a
> Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§
> Biophysical Journal Volume 94 March 2008 1826-1835
>
> If you have any questions, please contact me offline or phone.
>
> Also, the accuracy of the FRET eff. measurements could vary or
> be affected by the mounting media used - live cell FRET seems to
> be the best approach.
>
> Have a nice weekend,
>
> Vitaly
> 301-515-7833
>
>
>
>
>
> ________________________________
> From: John Oreopoulos <[hidden email]>
> To: [hidden email]
> Sent: Fri, April 16, 2010 12:01:32 PM
> Subject: Re: Mounting media for FRET experiments
>
> Just regular PBS solution. I have never tried a rigorous
> comparison of different media.
>
> John
>
> On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:
>
> > Thank you very much John, I found the paper you recommended
> and it is already proving useful. A question for you then -
> which media were you using for the live cells? What I mean is
> whether it was something like PBS or some specific media?
> >
> > ---------------------------------------------------
> > Yevgeniy Romin
> >
> > Digital Microscopist
> > Memorial Sloan-Kettering Cancer Center
> > Molecular Cytology Core Facility
> > 1275 York Ave. Box 333
> > New York, NY 10065
> > Tel.646-888-2186
> > Fax. 646-422-0640
> > ---------------------------------------------------
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of John Oreopoulos
> > Sent: Friday, April 16, 2010 11:30 AM
> > To: [hidden email]
> > Subject: Re: Mounting media for FRET experiments
> >
> > I have not used fixed specimens for FRET imaging (I have used
> the sensitized emission method on living cells), but I came
> across this paper a while ago which might be of interest to you:
> >
> > Rodighiero, S., et al., Fixation, mounting and sealing with
> nail polish of cell specimens lead to incorrect FRET
> measurements using acceptor photobleaching. Cellular Physiology
> and Biochemistry, 2008. 21(5-6): p. 489-498.
> >
> > John Oreopoulos
> >
> >
> > On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
> >
> >> Dear list
> >>
> >> During a recent FRET experiment a question came up about what
> mounting media should be used on fixed samples during FRET
> experiments and whether any one media is better than other for
> this particular type of experiment. From what's published it
> seems that people use all kinds of different media. Does
> anybody here have any idea about any specific media that would
> be best/give least interference with FRET results?
> >>
> >> Thanks a lot to all of you in advance,
> >>
> >> ---------------------------------------------------
> >> Yevgeniy Romin
> >>
> >> Digital Microscopist
> >> Memorial Sloan-Kettering Cancer Center
> >> Molecular Cytology Core Facility
> >> 1275 York Ave. Box 333
> >> New York, NY 10065
> >> Tel.646-888-2186
> >> Fax. 646-422-0640
> >> ---------------------------------------------------
> >>
> >>
> >>
> >>
> =====================================================================>>
> >> Please note that this e-mail and any files transmitted
> with it may be
> >> privileged, confidential, and protected from disclosure under
> >> applicable law. If the reader of this message is not the
> intended>> recipient, or an employee or agent responsible for
> delivering this
> >> message to the intended recipient, you are hereby notified
> that any
> >> reading, dissemination, distribution, copying, or other
> use of this
> >> communication or any of its attachments is strictly
> prohibited. If
> >> you have received this communication in error, please
> notify the
> >> sender immediately by replying to this message and
> deleting this
> >> message, any attachments, and all copies and backups from your
> >> computer.
> >>
> >>
>
>
>
>

Martin Spitaler

Re: Mounting media for FRET experiments

In reply to this post by Romin, Yevgeniy/Sloan Kettering Institute

Dear Yebgeniy,

you could also try CyGel (Biostatus) to immobilise your cells, it's liquid
on ice and solid at 37C, so you don't need to heat your cells. I haven't
tried it specifically for FRET, but we have used it for various other
experiments and it didn't seem to interfere with fluorescence.

- No commercial interest -

Martin
========================
Martin Spitaler, PhD

FILM - Facility for Imaging by Light Microscopy
- Facility Manager -
Sir Alexander Fleming Building, desk 401
Imperial College London / South Kensington
Exhibition Road
London SW7 2AZ
UK

Tel. +44-(0)20-759-42023
E-mail [hidden email]
Website: http://imperial.ac.uk/imagingfacility

On Sun, 18 Apr 2010 08:47:20 +1000, Cameron Nowell
<[hidden email]> wrote:

>Dear Yevgeniy,
>
>While i haven't had any experience imobilising cells for FRET we have had
great success in the past imobolising cells for ICS. We had a cell line that
expressed the receptor of interest but was round and didn't attache to
anything. We just put them in some 1% low melt temp agarose (made up in PBS)
and everything worked really well. Now we were only looking at the bleching
of one receptor, but any movement in the system would have been noticed in
the final analysis and there was none. I cant see a low percentage of
agarose interfereing with you FRET measurments.

>
>
>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: +61 3 9341 3155
>Mobile: +61422882700
>Fax: +61 3 9341 3104
>
>http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>
>
>________________________________
>
>From: Confocal Microscopy List on behalf of Yevgeniy Romin
>Sent: Sun 18/04/2010 1:49 AM
>To: [hidden email]
>Subject: Re: Mounting media for FRET experiments
>
>
>
>Thank you very much to everyone replying both on and off the list. Your

advice and the papers that were referred to were very helpful. FRET on live
samples would be the ideal choice, unfortunately in this case we are doing
FRET on white blood cells, which are very round and refuse to attach and sit
still, introducing motion artifacts into the FRET analysis. We are
currently working on immobilizing them, but there is a fear of matrigel or a
more viscous media introducing FRET measurement artifacts as well. Does
anyone have experience with doing FRET on cells that do not attach or
immobilizing them in a least invasive way?
>________________________________________
>From: Confocal Microscopy List [[hidden email]] On Behalf
Of Daniel Gitler [[hidden email]]
>Sent: Saturday, April 17, 2010 5:22 AM
>To: [hidden email]
>Subject: Re: Mounting media for FRET experiments
>
>Dear John and Yevgeniy,
>
>My experience when measuring FRET from the same samples before and after
fixation is that fixation itself will very much change the FRET efficiency
value that you measure (Cerulean and Venus in HEK cells, PBS for both).
Efficiency in the fixed samples was significantly lower. This should not
come as any big surprise, since the cellular environment around the
fluorophores will be very different.

>The possible reasons for the changes in FRET efficiency are very many...
>I also do most of my FRET imaging in live samples.
>
>Daniel
>
>----- Original Message -----
>From: Vitaly Boyko <[hidden email]>
>Date: Friday, April 16, 2010 19:36
>Subject: Re: Mounting media for FRET experiments
>To: [hidden email]
>
>> Dear John,
>> Dear Yevgeniy,
>>
>> Yes, PBS or specialized low serum (1-2%) media is broadly used
>> for live cell imaging under TIR illumination.
>>
>> I would like to bring your attention to two papers which are
>> relevant to single molecule fluorscence and/or low light live
>> cell imaging - I sent them separately to your e-mail addressess
>> as the LISTSERVER rejected my message due to the attachments. I
>> can also send the papers to anyone by request.
>>
>> 1. The Use of Protocatechuate Dioxygenase for Maintaining
>> Anaerobic Conditions in Biochemical ExperimentsPravin V. Patil
>> and David P. BallouDepartment of Biological Chemistry,
>> University of Michigan, Ann Arbor, Michigan 48109-0606
>> Analytical Biochemistry 286, 187-192 (2000)
>> 2. An Oxygen Scavenging System for Improvement of Dye Stability
>> in Single-Molecule Fluorescence ExperimentsColin Echeverri´a
>> Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§
>> Biophysical Journal Volume 94 March 2008 1826-1835
>>
>> If you have any questions, please contact me offline or phone.
>>
>> Also, the accuracy of the FRET eff. measurements could vary or
>> be affected by the mounting media used - live cell FRET seems to
>> be the best approach.
>>
>> Have a nice weekend,
>>
>> Vitaly
>> 301-515-7833
>>
>>
>>
>>
>>
>> ________________________________
>> From: John Oreopoulos <[hidden email]>
>> To: [hidden email]
>> Sent: Fri, April 16, 2010 12:01:32 PM
>> Subject: Re: Mounting media for FRET experiments
>>
>> Just regular PBS solution. I have never tried a rigorous
>> comparison of different media.
>>
>> John
>>
>> On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:
>>
>> > Thank you very much John, I found the paper you recommended
>> and it is already proving useful. A question for you then -
>> which media were you using for the live cells? What I mean is
>> whether it was something like PBS or some specific media?
>> >
>> > ---------------------------------------------------
>> > Yevgeniy Romin
>> >
>> > Digital Microscopist
>> > Memorial Sloan-Kettering Cancer Center
>> > Molecular Cytology Core Facility
>> > 1275 York Ave. Box 333
>> > New York, NY 10065
>> > Tel.646-888-2186
>> > Fax. 646-422-0640
>> > ---------------------------------------------------
>> >
>> > -----Original Message-----
>> > From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of John Oreopoulos
>> > Sent: Friday, April 16, 2010 11:30 AM
>> > To: [hidden email]
>> > Subject: Re: Mounting media for FRET experiments
>> >
>> > I have not used fixed specimens for FRET imaging (I have used
>> the sensitized emission method on living cells), but I came
>> across this paper a while ago which might be of interest to you:
>> >
>> > Rodighiero, S., et al., Fixation, mounting and sealing with
>> nail polish of cell specimens lead to incorrect FRET
>> measurements using acceptor photobleaching. Cellular Physiology
>> and Biochemistry, 2008. 21(5-6): p. 489-498.
>> >
>> > John Oreopoulos
>> >
>> >
>> > On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
>> >
>> >> Dear list
>> >>
>> >> During a recent FRET experiment a question came up about what
>> mounting media should be used on fixed samples during FRET
>> experiments and whether any one media is better than other for
>> this particular type of experiment. From what's published it
>> seems that people use all kinds of different media. Does
>> anybody here have any idea about any specific media that would
>> be best/give least interference with FRET results?
>> >>
>> >> Thanks a lot to all of you in advance,
>> >>
>> >> ---------------------------------------------------
>> >> Yevgeniy Romin
>> >>
>> >> Digital Microscopist
>> >> Memorial Sloan-Kettering Cancer Center
>> >> Molecular Cytology Core Facility
>> >> 1275 York Ave. Box 333
>> >> New York, NY 10065
>> >> Tel.646-888-2186
>> >> Fax. 646-422-0640
>> >> ---------------------------------------------------
>> >>
>> >>
>> >>
>> >>
>> =====================================================================>>
>> >> Please note that this e-mail and any files transmitted
>> with it may be
>> >> privileged, confidential, and protected from disclosure under
>> >> applicable law. If the reader of this message is not the
>> intended>> recipient, or an employee or agent responsible for
>> delivering this
>> >> message to the intended recipient, you are hereby notified
>> that any
>> >> reading, dissemination, distribution, copying, or other
>> use of this
>> >> communication or any of its attachments is strictly
>> prohibited. If
>> >> you have received this communication in error, please
>> notify the
>> >> sender immediately by replying to this message and
>> deleting this
>> >> message, any attachments, and all copies and backups from your
>> >> computer.
>> >>
>> >>
>>
>>
>>
>>
>
>Daniel Gitler, Ph.D.
>Department of Physiology and Neurobiology
>Faculty of Health Sciences
>Ben Gurion University of the Negev
>Beer-Sheva 84105
>Israel
>
>Tel: +972-8-6477345
>Cell: +972-54-2110100
>Fax: + 972-8-6477628
>http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/
>
>?

Charu Tanwar

Re: Mounting media for FRET experiments

In reply to this post by Romin, Yevgeniy/Sloan Kettering Institute

I have done FRET on Candida cells (live cells, not fixed) which are also round and small in size and refuse to attach onto the coverslip. I used Poly Lysine coated coverslip to adhere them on to the surface of the coverslip so that the movement is less. This you can do in your lab and these are commercially provided also.

After final washing with PBS, i mounted my cells with antifade reagent provided commercially ( it has some percentage of glycerol ). This has worked for me very well, although i can not say about WBC's.

Best

Charu

CHARU TANWAR
Imaging Specialist
Advanced Instrumentation Research Facility
Jawaharlal Nehru University
New Delhi 110067
India.

--- On Sat, 17/4/10, Yevgeniy Romin <[hidden email]> wrote:

From: Yevgeniy Romin <[hidden email]>
Subject: Re: Mounting media for FRET experiments
To: [hidden email]
Date: Saturday, 17 April, 2010, 9:19 PM

Thank you very much to everyone replying both on and off the list. Your advice and the papers that were referred to were very helpful. FRET on live samples would be the ideal choice, unfortunately in this case we are doing FRET on white blood cells, which are very round and refuse to attach and sit still, introducing motion artifacts into the FRET analysis. We are currently working on immobilizing them, but there is a fear of matrigel or a more viscous media introducing FRET measurement artifacts as well. Does anyone have experience with doing FRET on cells that do not attach or immobilizing them in a least invasive way?
________________________________________
From: Confocal Microscopy List [CONFOCALMICROSCOPY@...] On Behalf Of Daniel Gitler [gitler@...]
Sent: Saturday, April 17, 2010 5:22 AM
To: CONFOCALMICROSCOPY@...
Subject: Re: Mounting media for FRET experiments

Dear John and Yevgeniy,

My experience when measuring FRET from the same samples before and after fixation is that fixation itself will very much change the FRET efficiency value that you measure (Cerulean and Venus in HEK cells, PBS for both). Efficiency in the fixed samples was significantly lower. This should not come as any big surprise, since the cellular environment around the fluorophores will be very different.
The possible reasons for the changes in FRET efficiency are very many...
I also do most of my FRET imaging in live samples.

Daniel

----- Original Message -----
From: Vitaly Boyko <boyko.vitaly@...>
Date: Friday, April 16, 2010 19:36
Subject: Re: Mounting media for FRET experiments
To: CONFOCALMICROSCOPY@...

> Dear John,
> Dear Yevgeniy,
>
> Yes, PBS or specialized low serum (1-2%) media is broadly used
> for live cell imaging under TIR illumination.
>
> I would like to bring your attention to two papers which are
> relevant to single molecule fluorscence and/or low light live
> cell imaging - I sent them separately to your e-mail addressess
> as the LISTSERVER rejected my message due to the attachments. I
> can also send the papers to anyone by request.
>
> 1. The Use of Protocatechuate Dioxygenase for Maintaining
> Anaerobic Conditions in Biochemical ExperimentsPravin V. Patil
> and David P. BallouDepartment of Biological Chemistry,
> University of Michigan, Ann Arbor, Michigan 48109-0606
> Analytical Biochemistry 286, 187–192 (2000)
> 2. An Oxygen Scavenging System for Improvement of Dye Stability
> in Single-Molecule Fluorescence ExperimentsColin Echeverrı´a
> Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§
> Biophysical Journal Volume 94 March 2008 1826–1835
>
> If you have any questions, please contact me offline or phone.
>
> Also, the accuracy of the FRET eff. measurements could vary or
> be affected by the mounting media used - live cell FRET seems to
> be the best approach.
>
> Have a nice weekend,
>
> Vitaly
> 301-515-7833
>
>
>
>
>
> ________________________________
> From: John Oreopoulos <john.oreopoulos@...>
> To: CONFOCALMICROSCOPY@...
> Sent: Fri, April 16, 2010 12:01:32 PM
> Subject: Re: Mounting media for FRET experiments
>
> Just regular PBS solution. I have never tried a rigorous
> comparison of different media.
>
> John
>
> On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:
>
> > Thank you very much John, I found the paper you recommended
> and it is already proving useful. A question for you then -
> which media were you using for the live cells? What I mean is
> whether it was something like PBS or some specific media?
> >
> > ---------------------------------------------------
> > Yevgeniy Romin
> >
> > Digital Microscopist
> > Memorial Sloan-Kettering Cancer Center
> > Molecular Cytology Core Facility
> > 1275 York Ave. Box 333
> > New York, NY 10065
> > Tel.646-888-2186
> > Fax. 646-422-0640
> > ---------------------------------------------------
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:CONFOCALMICROSCOPY@...] On Behalf Of John Oreopoulos
> > Sent: Friday, April 16, 2010 11:30 AM
> > To: CONFOCALMICROSCOPY@...
> > Subject: Re: Mounting media for FRET experiments
> >
> > I have not used fixed specimens for FRET imaging (I have used
> the sensitized emission method on living cells), but I came
> across this paper a while ago which might be of interest to you:
> >
> > Rodighiero, S., et al., Fixation, mounting and sealing with
> nail polish of cell specimens lead to incorrect FRET
> measurements using acceptor photobleaching. Cellular Physiology
> and Biochemistry, 2008. 21(5-6): p. 489-498.
> >
> > John Oreopoulos
> >
> >
> > On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
> >
> >> Dear list
> >>
> >> During a recent FRET experiment a question came up about what
> mounting media should be used on fixed samples during FRET
> experiments and whether any one media is better than other for
> this particular type of experiment. From what's published it
> seems that people use all kinds of different media. Does
> anybody here have any idea about any specific media that would
> be best/give least interference with FRET results?
> >>
> >> Thanks a lot to all of you in advance,
> >>
> >> ---------------------------------------------------
> >> Yevgeniy Romin
> >>
> >> Digital Microscopist
> >> Memorial Sloan-Kettering Cancer Center
> >> Molecular Cytology Core Facility
> >> 1275 York Ave. Box 333
> >> New York, NY 10065
> >> Tel.646-888-2186
> >> Fax. 646-422-0640
> >> ---------------------------------------------------
> >>
> >>
> >>
> >>
> =====================================================================>>
> >> Please note that this e-mail and any files transmitted
> with it may be
> >> privileged, confidential, and protected from disclosure under
> >> applicable law. If the reader of this message is not the
> intended>> recipient, or an employee or agent responsible for
> delivering this
> >> message to the intended recipient, you are hereby notified
> that any
> >> reading, dissemination, distribution, copying, or other
> use of this
> >> communication or any of its attachments is strictly
> prohibited. If
> >> you have received this communication in error, please
> notify the
> >> sender immediately by replying to this message and
> deleting this
> >> message, any attachments, and all copies and backups from your
> >> computer.
> >>
> >>
>
>
>
>

Daniel Gitler, Ph.D.
Department of Physiology and Neurobiology
Faculty of Health Sciences
Ben Gurion University of the Negev
Beer-Sheva 84105
Israel

Tel: +972-8-6477345
Cell: +972-54-2110100
Fax: + 972-8-6477628
http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/

‎

Janne Hyötylä

Re: Mounting media for FRET experiments

This article might also provide further ideas for cell adhesion substrates:
Hwang, D.S., Sim, S.B. & Cha, H.J. Cell adhesion biomaterial based on
mussel adhesive protein fused with RGD peptide. Biomaterials 28,
4039-46(2007).

Cheers
Janne

On Tue, 20 Apr 2010 10:27:27 +0200, charu tanwar
<[hidden email]> wrote:

> I have done FRET on Candida cells (live cells, not fixed) which are also
> round and small in size and refuse to attach onto the coverslip. I used
> Poly Lysine coated coverslip to adhere them on to the surface of the
> coverslip so that the movement is less. This you can do in your lab and
> these are commercially provided also.
> After final washing with PBS, i mounted my cells with antifade reagent
> provided commercially ( it has some percentage of glycerol ). This has
> worked for me very well, although i can not say about WBC's.
> Best
> Charu
>
> CHARU TANWAR
> Imaging Specialist
> Advanced Instrumentation Research Facility
> Jawaharlal Nehru University
> New Delhi 110067
> India.
>
> --- On Sat, 17/4/10, Yevgeniy Romin <[hidden email]> wrote:
>
>
> From: Yevgeniy Romin <[hidden email]>
> Subject: Re: Mounting media for FRET experiments
> To: [hidden email]
> Date: Saturday, 17 April, 2010, 9:19 PM
>
>
> Thank you very much to everyone replying both on and off the list. Your
> advice and the papers that were referred to were very helpful. FRET on
> live samples would be the ideal choice, unfortunately in this case we
> are doing FRET on white blood cells, which are very round and refuse to
> attach and sit still, introducing motion artifacts into the FRET
> analysis. We are currently working on immobilizing them, but there is a
> fear of matrigel or a more viscous media introducing FRET measurement
> artifacts as well. Does anyone have experience with doing FRET on cells
> that do not attach or immobilizing them in a least invasive way?
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On
> Behalf Of Daniel Gitler [[hidden email]]
> Sent: Saturday, April 17, 2010 5:22 AM
> To: [hidden email]
> Subject: Re: Mounting media for FRET experiments
>
> Dear John and Yevgeniy,
>
> My experience when measuring FRET from the same samples before and after
> fixation is that fixation itself will very much change the FRET
> efficiency value that you measure (Cerulean and Venus in HEK cells, PBS
> for both). Efficiency in the fixed samples was significantly lower. This
> should not come as any big surprise, since the cellular environment
> around the fluorophores will be very different.
> The possible reasons for the changes in FRET efficiency are very many...
> I also do most of my FRET imaging in live samples.
>
> Daniel
>
> ----- Original Message -----
> From: Vitaly Boyko <[hidden email]>
> Date: Friday, April 16, 2010 19:36
> Subject: Re: Mounting media for FRET experiments
> To: [hidden email]
>
>> Dear John,
>> Dear Yevgeniy,
>>
>> Yes, PBS or specialized low serum (1-2%) media is broadly used
>> for live cell imaging under TIR illumination.
>>
>> I would like to bring your attention to two papers which are
>> relevant to single molecule fluorscence and/or low light live
>> cell imaging - I sent them separately to your e-mail addressess
>> as the LISTSERVER rejected my message due to the attachments. I
>> can also send the papers to anyone by request.
>>
>> 1. The Use of Protocatechuate Dioxygenase for Maintaining
>> Anaerobic Conditions in Biochemical ExperimentsPravin V. Patil
>> and David P. BallouDepartment of Biological Chemistry,
>> University of Michigan, Ann Arbor, Michigan 48109-0606
>> Analytical Biochemistry 286, 187–192 (2000)
>> 2. An Oxygen Scavenging System for Improvement of Dye Stability
>> in Single-Molecule Fluorescence ExperimentsColin Echeverrı´a
>> Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§
>> Biophysical Journal Volume 94 March 2008 1826–1835
>>
>> If you have any questions, please contact me offline or phone.
>>
>> Also, the accuracy of the FRET eff. measurements could vary or
>> be affected by the mounting media used - live cell FRET seems to
>> be the best approach.
>>
>> Have a nice weekend,
>>
>> Vitaly
>> 301-515-7833
>>
>>
>>
>>
>>
>> ________________________________
>> From: John Oreopoulos <[hidden email]>
>> To: [hidden email]
>> Sent: Fri, April 16, 2010 12:01:32 PM
>> Subject: Re: Mounting media for FRET experiments
>>
>> Just regular PBS solution. I have never tried a rigorous
>> comparison of different media.
>>
>> John
>>
>> On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:
>>
>> > Thank you very much John, I found the paper you recommended
>> and it is already proving useful. A question for you then -
>> which media were you using for the live cells? What I mean is
>> whether it was something like PBS or some specific media?
>> >
>> > ---------------------------------------------------
>> > Yevgeniy Romin
>> >
>> > Digital Microscopist
>> > Memorial Sloan-Kettering Cancer Center
>> > Molecular Cytology Core Facility
>> > 1275 York Ave. Box 333
>> > New York, NY 10065
>> > Tel.646-888-2186
>> > Fax. 646-422-0640
>> > ---------------------------------------------------
>> >
>> > -----Original Message-----
>> > From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of John Oreopoulos
>> > Sent: Friday, April 16, 2010 11:30 AM
>> > To: [hidden email]
>> > Subject: Re: Mounting media for FRET experiments
>> >
>> > I have not used fixed specimens for FRET imaging (I have used
>> the sensitized emission method on living cells), but I came
>> across this paper a while ago which might be of interest to you:
>> >
>> > Rodighiero, S., et al., Fixation, mounting and sealing with
>> nail polish of cell specimens lead to incorrect FRET
>> measurements using acceptor photobleaching. Cellular Physiology
>> and Biochemistry, 2008. 21(5-6): p. 489-498.
>> >
>> > John Oreopoulos
>> >
>> >
>> > On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
>> >
>> >> Dear list
>> >>
>> >> During a recent FRET experiment a question came up about what
>> mounting media should be used on fixed samples during FRET
>> experiments and whether any one media is better than other for
>> this particular type of experiment. From what's published it
>> seems that people use all kinds of different media. Does
>> anybody here have any idea about any specific media that would
>> be best/give least interference with FRET results?
>> >>
>> >> Thanks a lot to all of you in advance,
>> >>
>> >> ---------------------------------------------------
>> >> Yevgeniy Romin
>> >>
>> >> Digital Microscopist
>> >> Memorial Sloan-Kettering Cancer Center
>> >> Molecular Cytology Core Facility
>> >> 1275 York Ave. Box 333
>> >> New York, NY 10065
>> >> Tel.646-888-2186
>> >> Fax. 646-422-0640
>> >> ---------------------------------------------------
>> >>
>> >>
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>
> Daniel Gitler, Ph.D.
> Department of Physiology and Neurobiology
> Faculty of Health Sciences
> Ben Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> Tel: +972-8-6477345
> Cell: +972-54-2110100
> Fax: + 972-8-6477628
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/
>
> ‎
>
>

--
Janne Hyötylä
Biozentrum and the Swiss Nanoscience Institute
University of Basel
Klingelbergstr. 50/70
CH-4056 Basel
Switzerland

tel: +41 61 267 2082
e-mail: [hidden email]