Researcher/postdoc National Imaging Platform-Oslo

[Oddmund Bakke]

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

POSITION AS POSTDOC/RESEARCH FELLOW (s) available at
National Subcellular Imaging Platform, NorMIC-UIO, DEPARTMENT OF
MOLECULAR BIOSCIENCES, Faculty of Mathematics and Natural Sciences,
University of Oslo, Norway


The position (s) is available for a period of three years (possible prolongation)
Start date:  January, 2012  or as soon as possible.

The national imaging platform has received a 20 mill instrument grant from
the Research Council of Norway and will expand and get new instruments for
subcellular imaging. We are looking for a postdoc/researcher with a strong
background in cell biology and imaging. The position is financed by the Faculty
of Mathematics and Natural Sciences, University of Oslo for 3 years.

The aim of the national imaging service platform is to offer state-of-the-art
imaging techniques and expertise within subcellular imaging to local, national
and international research groups. The focus is on live cell confocal
microscopy, high throughput imaging and micromanipulation of cells. The
platform offers today live cell imaging techniques such as , FRAP, FRET and
photoactivation. With the new equipment we will expand the techniques with
TIRF, FLIM, micromanipulation, microinjection and superresolution,. We are
mainly working with tissue culture cell lines, primary cells, immune cells and
single cell organisms. The different techniques will vary depending on the
development of the various projects at the platform. The equipment also
allows working with cell types and single cell organism living at different
temperatures. The candidate is expected to participate in advanced imaging
projects that require independent scientific work and may run independent
projects. The position require that the candidate gives support to users and
teaches in user courses. The candidate should also be involved in developing
and establishing new imaging protocols depending on the project in question.

The platform at IMBV is part of the NorMIC national FUGE consortium and is
closely connected to its partner at the Radium Hospital (group of prof Harald
Stenmark). The imaging platform also offer electron microscopy techniques
(CLEM-correlative light and electron microscopy and immuno-EM. The platform
is furthermore a host for Eurobioimaging Proof of Concept studies 2012 (see
www.eurobioimaging.eu).
Platform page:
www.mn.uio.no/imbv/english/research/about/infrastructure/imaging

The head of the platform is Professor Oddmund Bakke. The imaging platform
has at present one temporary postdoc position and two permanent advanced
technical engineers. The expertise within imaging also depends on the imaging
project in the Bakke research group, which consist of 4 master students, 2
PhD students, and 4 postdocs. The Bakke group is a member of the CoE
(Centre of Excellence) “Centre for Immune Regulation” (CIR (www.cir.uio.no)
and the focus of the group is on membrane trafficking in immune cells.

We are looking for highly motivated and competent candidates with
experimental and theoretical background in cell biology, immunology,
biochemistry, bioinformatics and/or biophysics. Successful candidates should
have hands-on experience with technical instruments within
biology/biotechnology and be able to handle biological specimens such as cells
and tissue. Previous experience from imaging is absolutely an advantage but
not a prerequisite as he/she will be part of an active imaging environment
with courses/training program.

The position requires a PhD or equivalent within cell biology, biochemistry,
immunology, biophysics or bioinformatics with a strong element of
instrumental techniques. A second position at the platform will also be
announced and suitable candidates not hired will be asked if they will apply for
this position. A PhD may not be required for the second position.

The letter of application should include curriculum vitae with a list of published
and unpublished works with a statement summarizing the applicant’s
scientific work and interests in addition to certified copies of certificates.

For further information please contact:
professor Oddmund Bakke, phone [phone] or : + 47 22 855787
e-mail: [hidden email]

The position is available for a period of three years

Likely deadline for application:  Desember 19th 2011.

REF.NO.  ? See notice when available at
www.uio.no/om/jobb/ledige-stillinehger/

Applicant should submit a letter of application (marked with REF.No. 2011/??),
CV, a list of published and unpublished works and 3 copies of published papers
not available in medline. Three sets of copies of educational transcripts and
diplomas should also be submitted.

Applications should be sent to: Faculty of Mathematics and Natural Sciences,
attn. Senior Executive Officer Bente Schjoldager, P. O. Box 1032 Blindern, N-
0315 Oslo, Norway.

The application may also be submitted by e-mail to [hidden email]
NB!  REMEMBER TO ALWAYS PUT ON REF.NO. IN “SUBJECT”.

[Straatman, Kees (Dr.)]

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

Dear list members,

We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?

Thanks

Kees

Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester

http://www.le.ac.uk/biochem/microscopy/home.html

[Guy Cox-2]

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

I'm a bit lost by this.  As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization.  I may have misunderstood your point - if so, please clarify.  My brain is probably slowing down with age!

                               Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To: [hidden email]
Subject: FRET-FLIM question

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

Dear list members,

We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?

Thanks

Kees

Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester

http://www.le.ac.uk/biochem/microscopy/home.html

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11

[Straatman, Kees (Dr.)]

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

Hi Guy,

Sorry, will try to explain it a little more. In the publication they found an increased FRET efficiency when an unlabelled binding partner is added to the cells and they argued that this is caused by an increase of dimerisation of their CFP and YFP labelled proteins. As FRET-FLIM is independent of concentration I would expect that there is a structural change within the dimer what brings the donor and acceptor closer together resulting in an increase of FRET efficiency. Using a Bimolecular fluorescence complementation (BiFC) system we find less signal when we add this binding partner. So I was wondering if it is possible that less dimer is formed but the dimer that is formed has a changed configuration and gives a higher FRET-FLIM signal. But as I don't know enough about FLIM I am not sure if you can see a difference in number of molecules that result in a FRET-FLIM signal by using for example the time it takes to collect a signal.

Kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: 17 November 2011 12:32
To: [hidden email]
Subject: Re: FRET-FLIM question

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

I'm a bit lost by this.  As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization.  I may have misunderstood your point - if so, please clarify.  My brain is probably slowing down with age!

                               Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To: [hidden email]
Subject: FRET-FLIM question

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

Dear list members,

We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?

Thanks

Kees

Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester

http://www.le.ac.uk/biochem/microscopy/home.html

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11

[Emmanuel Gustin]

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

It depends on what FLIM system they use. If they were able to acquire enough information to *reliably* fit the donor fluorescence data to a bi-exponential:

        A exp(-(t-t0)/tau1) + B exp(-(t-t0)/tau2)

Where tau1 is the lifetime of the donor in a FRET couple and tau2 is the lifetime of the donor with "remote" acceptors, then the ratio of areas below the A and B curves would be a good measure for efficiency of dimerisation.

But in many FLIM experiments, as far as I know, the data is too rough to do that, and then the best option is to "fit" a single exponential. That gives you an "pragmatic" lifetime estimate tau that reflects a mixture of donor-acceptance distance and dimerisation efficiency. It actually depends on all of the four above parameters.

Best Regards,

Emmanuel


--
 Emmanuel Gustin,    Tel. [phone],    e-mail: [hidden email]  


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: donderdag 17 november 2011 13:32
To: [hidden email]
Subject: Re: FRET-FLIM question

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

I'm a bit lost by this.  As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization.  I may have misunderstood your point - if so, please clarify.  My brain is probably slowing down with age!

                               Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To: [hidden email]
Subject: FRET-FLIM question

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

Dear list members,

We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?

Thanks

Kees

Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester

http://www.le.ac.uk/biochem/microscopy/home.html

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11

[Tim Feinstein-2]

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

Hi Kees,

FLIM data reports the change in the amount of donor energy transferred to the acceptor either per cell or per pixel.   One source of your problem could be that energy transfer always convolves the number of interacting proteins with the distance and alignment between donor and acceptor, both of which can change in a single experiment.   Most FRET studies try to simplify this question by assuming away the complexity - either dimers form of they do not (as in your colleague's argument), the complex is fixed and you measure its conformational change (e.g., most modern biosensors) or no binding is assumed and you are simply measuring the concentration of the two fluorophores (e.g., similar to fluorescence dequenching in liposome fusion).  You are asking whether the complexes can decrease in number while the remaining complexes change in conformation to bring the fluorophores much closer together.  This is possible in theory, but it would mean that the initial FRET had to be quite low and the loss of dimerization would have to be modest.  That kind of question is best tested using an independent measure of dimerization - you could try a co-IP, or else immobilize one binding partner with an antibody or cytoskeleton cross-linking domain and measure the mobility of the other using FRAP.  

Now I am confused by your description of BIFC data.  Bimolecular complementation is an irreversible reaction.  By 'goes down' do you mean that the rate of BIFC complex formation decreases, or do you really see a decrease in BIFC fluorescence when you add the third binding partner?  I would be concerned if the second case was true.  

cheers,


TF

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Nov 17, 2011, at 7:51 AM, Straatman, Kees R. (Dr.) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Guy,
>
> Sorry, will try to explain it a little more. In the publication they found an increased FRET efficiency when an unlabelled binding partner is added to the cells and they argued that this is caused by an increase of dimerisation of their CFP and YFP labelled proteins. As FRET-FLIM is independent of concentration I would expect that there is a structural change within the dimer what brings the donor and acceptor closer together resulting in an increase of FRET efficiency. Using a Bimolecular fluorescence complementation (BiFC) system we find less signal when we add this binding partner. So I was wondering if it is possible that less dimer is formed but the dimer that is formed has a changed configuration and gives a higher FRET-FLIM signal. But as I don't know enough about FLIM I am not sure if you can see a difference in number of molecules that result in a FRET-FLIM signal by using for example the time it takes to collect a signal.
>
> Kees
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
> Sent: 17 November 2011 12:32
> To: [hidden email]
> Subject: Re: FRET-FLIM question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm a bit lost by this.  As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization.  I may have misunderstood your point - if so, please clarify.  My brain is probably slowing down with age!
>
>                               Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
> Sent: Thursday, 17 November 2011 9:34 PM
> To: [hidden email]
> Subject: FRET-FLIM question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list members,
>
> We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?
>
> Thanks
>
> Kees
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Centre for Core Biotechnology Services
> College of Medicine, Biological Sciences and Psychology
> University of Leicester
>
> http://www.le.ac.uk/biochem/microscopy/home.html
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11

[Vitaly Boyko]

I am a bit lost as well here.

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

Hi Kees,

I am a bit lost as well here.

If CFP-YPF tagged proteins are "diluted" with untagged proteins and increased FRET signal is reported, it might also point out to a more complex situation such as self-quenching of acceptor-by-acceptor and possible oligomerization (rather than dimerization of CFP-YFP tagged molecules). I would assume that CFP and/or YFP, being dimers themselves, could also stabilize any proteins that are expected to form a dimer, especially if local concentration of CFP or YFP is ca. 100-200uM or even less!!! 
Monomeric CeFP or YFP might help, only a bit I would guess. Though I did lots of experiments in that direction, I did not have enough time to analyze it comprehensively. However, I have also not seen careful comparative FRET study of different FRET pairs (e.g. CeFP or YFP with or without A206K vs CeFP (YFP) with all three A206K, L221K, F223R monomeric mutations, etc.) with good and convincing statistics and accurate quantitative analysis on a voxel-by-voxel basis (or 3x3x3 VOIs).

It would be good to know what was the imaging setup used in the FRET experiment including time post transfection. I have seen lots of strange things if images were taken later that 18 hours post transfection in HELA cells with less than 1 ug of total DNA per 35mm dish.

Thus, statement of FLIM-FRET independence of concentration is only true in theory (or with highly soluble donors and acceptors in a test tube) in an environment that is free of oligomerization or non-specific aggregation of CFP/YFP tagged proteins (CFP/YFP are hydrophobic and sticky to membranes at high concentrations). And in real experiment it could be a mixture of true dimers and artefactual oligomers. And it might be difficult to separate true dimers from oligomers withing a single 3x3x3 VOI.

I can also tell you that in BiFC experiment, when mCherry was fused to split venusYFP, very strong BiFC signal (reconstituted venusYFP) has been recorded already at 12 hours post transfection pointing out either to weakness of BiFC system or non-monomeric nature of "pseudo"???-monomeric mCherry.

Thus, "clean" quantitative imaging is still a challenging task. There are some solutions to it - one of them is here - every Research Institution should have an established PI with strong background in Fundamental Biophysics, possibly with a degree in Physics rather than Biology. Promoting broadly integrated inter disciplinary environment is a priority that politicians are not aware of as they seem to be very busy with cleaning up the global economic "mess".

On an optimistic note -  with new tech developments, imaging and accurate image analysis gets better very fast.

Vitaly

  


________________________________
From: "Straatman, Kees R. (Dr.)" <[hidden email]>
To: [hidden email]
Sent: Thursday, November 17, 2011 7:51 AM
Subject: Re: FRET-FLIM question

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

Hi Guy,

Sorry, will try to explain it a little more. In the publication they found an increased FRET efficiency when an unlabelled binding partner is added to the cells and they argued that this is caused by an increase of dimerisation of their CFP and YFP labelled proteins. As FRET-FLIM is independent of concentration I would expect that there is a structural change within the dimer what brings the donor and acceptor closer together resulting in an increase of FRET efficiency. Using a Bimolecular fluorescence complementation (BiFC) system we find less signal when we add this binding partner. So I was wondering if it is possible that less dimer is formed but the dimer that is formed has a changed configuration and gives a higher FRET-FLIM signal. But as I don't know enough about FLIM I am not sure if you can see a difference in number of molecules that result in a FRET-FLIM signal by using for example the time it takes to collect a signal.

Kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: 17 November 2011 12:32
To: [hidden email]
Subject: Re: FRET-FLIM question

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

I'm a bit lost by this.  As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization.  I may have misunderstood your point - if so, please clarify.  My brain is probably slowing down with age!

                               Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To: [hidden email]
Subject: FRET-FLIM question

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

Dear list members,

We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?

Thanks

Kees

Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester

http://www.le.ac.uk/biochem/microscopy/home.html

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11
Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To: [hidden email]
Subject: FRET-FLIM question

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

Dear list members,

We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?

Thanks

Kees

Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester

http://www.le.ac.uk/biochem/microscopy/home.html

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11

[Deepak nair]

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

Hi All

I am sorry a bit of understanding/misunderstanding from my part. I thought
that the concentration dependence of life times depend on how you do your
analysis (Like Emmanuel suggested). If the analysis is based on mean or
average lifetimes, it is concentration dependent since it averages
fractional contributions of multiple lifetimes in the decay.On the
otherhand If you can find out the individual components (wether bi (for
GFP) or multiexpoential (for CFP)) of the decay, it is concentration
independent since it gives out the unaveraged lifetimes of individual
species.

Can someone correct me if I got it wrong...

Best
Deepak

On Thu, Nov 17, 2011 at 1:58 PM, Gustin, Emmanuel [JRDBE] <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It depends on what FLIM system they use. If they were able to acquire
> enough information to *reliably* fit the donor fluorescence data to a
> bi-exponential:
>
>        A exp(-(t-t0)/tau1) + B exp(-(t-t0)/tau2)
>
> Where tau1 is the lifetime of the donor in a FRET couple and tau2 is the
> lifetime of the donor with "remote" acceptors, then the ratio of areas
> below the A and B curves would be a good measure for efficiency of
> dimerisation.
>
> But in many FLIM experiments, as far as I know, the data is too rough to
> do that, and then the best option is to "fit" a single exponential. That
> gives you an "pragmatic" lifetime estimate tau that reflects a mixture of
> donor-acceptance distance and dimerisation efficiency. It actually depends
> on all of the four above parameters.
>
> Best Regards,
>
> Emmanuel
>
>
> --
>  Emmanuel Gustin,    Tel. [phone],    e-mail: [hidden email]
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Guy Cox
> Sent: donderdag 17 november 2011 13:32
> To: [hidden email]
> Subject: Re: FRET-FLIM question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm a bit lost by this.  As you say, donor lifetime reduction caused by
> FRET is independent of concentration (so long as there is no external
> constraint on dimer formation), but since dimer formation is typically what
> brings the FRET partners into close proximity, I cannot see how you can
> expect to have increased FRET efficiency with less dimerization.  I may
> have misunderstood your point - if so, please clarify.  My brain is
> probably slowing down with age!
>
>                               Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Straatman, Kees R. (Dr.)
> Sent: Thursday, 17 November 2011 9:34 PM
> To: [hidden email]
> Subject: FRET-FLIM question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list members,
>
> We have a discussion about FRET-FLIM at the moment and somebody has
> published that they see an increase in FRET efficiency and argues that this
> is because there is more dimerisation between the two proteins they are
> interested in. Now, I thought that FRET-FLIM is independent from the
> concentration of the fluorochromes and that the increase in FRET efficiency
> is caused by a change in distance between the 2 reporter FP (CFP and YFP)
> and it would be possible to have less dimerisation of the proteins but an
> increase in FRET efficiency. Is this correct?
>
> Thanks
>
> Kees
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Centre for Core Biotechnology Services
> College of Medicine, Biological Sciences and Psychology
> University of Leicester
>
> http://www.le.ac.uk/biochem/microscopy/home.html
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11
>

--

[Straatman, Kees (Dr.)]

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

Dear Timothy,

Thanks for the answer. By goes down I meant that we get a lower ratio signal using the third binding partner than in the control cells without the third binding partner. We do a transfection using plasmids of the BIFC partners and a RFP and we ratio the BIFC signal against the RFP signal. When we add to this mix the third binging partner this ratio is lower.

Kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tim Feinstein
Sent: 17 November 2011 15:14
To: [hidden email]
Subject: Re: FRET-FLIM question

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

Hi Kees,

FLIM data reports the change in the amount of donor energy transferred to the acceptor either per cell or per pixel.   One source of your problem could be that energy transfer always convolves the number of interacting proteins with the distance and alignment between donor and acceptor, both of which can change in a single experiment.   Most FRET studies try to simplify this question by assuming away the complexity - either dimers form of they do not (as in your colleague's argument), the complex is fixed and you measure its conformational change (e.g., most modern biosensors) or no binding is assumed and you are simply measuring the concentration of the two fluorophores (e.g., similar to fluorescence dequenching in liposome fusion).  You are asking whether the complexes can decrease in number while the remaining complexes change in conformation to bring the fluorophores much closer together.  This is possible in theory, but it would mean that the initial FRET had to be quite low and the loss of dimerization would have to be modest.  That kind of question is best tested using an independent measure of dimerization - you could try a co-IP, or else immobilize one binding partner with an antibody or cytoskeleton cross-linking domain and measure the mobility of the other using FRAP.  

Now I am confused by your description of BIFC data.  Bimolecular complementation is an irreversible reaction.  By 'goes down' do you mean that the rate of BIFC complex formation decreases, or do you really see a decrease in BIFC fluorescence when you add the third binding partner?  I would be concerned if the second case was true.  

cheers,


TF

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Nov 17, 2011, at 7:51 AM, Straatman, Kees R. (Dr.) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Guy,
>
> Sorry, will try to explain it a little more. In the publication they found an increased FRET efficiency when an unlabelled binding partner is added to the cells and they argued that this is caused by an increase of dimerisation of their CFP and YFP labelled proteins. As FRET-FLIM is independent of concentration I would expect that there is a structural change within the dimer what brings the donor and acceptor closer together resulting in an increase of FRET efficiency. Using a Bimolecular fluorescence complementation (BiFC) system we find less signal when we add this binding partner. So I was wondering if it is possible that less dimer is formed but the dimer that is formed has a changed configuration and gives a higher FRET-FLIM signal. But as I don't know enough about FLIM I am not sure if you can see a difference in number of molecules that result in a FRET-FLIM signal by using for example the time it takes to collect a signal.
>
> Kees
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
> Sent: 17 November 2011 12:32
> To: [hidden email]
> Subject: Re: FRET-FLIM question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm a bit lost by this.  As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization.  I may have misunderstood your point - if so, please clarify.  My brain is probably slowing down with age!
>
>                               Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
> Sent: Thursday, 17 November 2011 9:34 PM
> To: [hidden email]
> Subject: FRET-FLIM question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list members,
>
> We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?
>
> Thanks
>
> Kees
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Centre for Core Biotechnology Services
> College of Medicine, Biological Sciences and Psychology
> University of Leicester
>
> http://www.le.ac.uk/biochem/microscopy/home.html
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11