Confocal Microscopy List

mKO with 2-Photon

Classic

List

Threaded

12 messages Options

Jamie Hayden

mKO with 2-Photon

I have a researcher trying to plan future experiments with fluorescent proteins and we are trying to determine what the best options would be based on our equipment set. They are considering mCherry vs mKO and are leaning toward mKO (I think there is an mKO2 now) because the literature shows it to be brighter and more stable. They would like to do in vivo work with 2 photon (we have a Prairie Ultima 2 with a Coherent Chameleon XR laser: 720 - 980nm). Ideally, they would also like to do single photon confocal, but we are limited to a 568 laser at the moment (hoping to upgrade soon), which seems to be just a bit too high for mKO.

Has anyone worked with these proteins (especially in vivo) who could give me an idea of the excitation peaks with standard confocal and 2-P? Characterization of mCherry is pretty good, but there is very little to go on with the mKO. Any help pointing to some literature would be most appreciated as well.

James E. Hayden, RBP, FBCA

Manager

Microscopy Core Facility

The Wistar Institute

3601 Spruce St.

Philadelphia, PA 19104

(215)898-3887

[hidden email]

Haberman, Ann

Re: mKO with 2-Photon

Hi Jamie,

Is mKO the same as Kate? I afraid that I don't have any experience with mKO, but do know that you will not be able to image mCherry with your laser. mCherry requires wavelengths longer than 1,000 nm.

I do have a dsRed variant to suggest though! The dsRed express variant sold by Clontech is very bright and easy to excite with both a two-photon laser and the 568 laser. It is compatible with excitation of GFP and CFP at a wavelength of 865 nm with the 2p laser. I would definitely suggest that they buy this fluorophore instead!

There is a strain of mice that expresses this ubiquitously sold by Jax made by the Nagy lab. I use it all the time and love it. The fluorescent protein itself was engineered by the Glick lab (chicago I think)

best,

Ann

I have a researcher trying to plan future experiments with fluorescent proteins and we are trying to determine what the best options would be based on our equipment set. They are considering mCherry vs mKO and are leaning toward mKO (I think there is an mKO2 now) because the literature shows it to be brighter and more stable. They would like to do in vivo work with 2 photon (we have a Prairie Ultima 2 with a Coherent Chameleon XR laser: 720 - 980nm). Ideally, they would also like to do single photon confocal, but we are limited to a 568 laser at the moment (hoping to upgrade soon), which seems to be just a bit too high for mKO.

Has anyone worked with these proteins (especially in vivo) who could give me an idea of the excitation peaks with standard confocal and 2-P? Characterization of mCherry is pretty good, but there is very little to go on with the mKO. Any help pointing to some literature would be most appreciated as well.

James E. Hayden, RBP, FBCA

Manager

Microscopy Core Facility

The Wistar Institute

3601 Spruce St.

Philadelphia, PA 19104

(215)898-3887

[hidden email]

--

Ann Haberman, PhD
Department of Laboratory Medicine
Yale University School of Medicine
1 Gilbert St.
TAC S541
New Haven, CT 06510

203-785-7349
203-785-5415 (fax)
[hidden email]

Craig Brideau

Re: mKO with 2-Photon

In reply to this post by Jamie Hayden

Another trick is to frequency-double your Ti:Saph for single photon
confocal work. You need a KTP crystal or the like, costing about
$1000. It will halve the wavelength of your laser, whatever your
laser is tuned to. You have to rotate the crystal to match the
frequency, but it is pretty easy to do with a basic rotation stage.

Craig

On Thu, Nov 12, 2009 at 11:22 AM, Ann Haberman <[hidden email]> wrote:

> Hi Jamie,
> Is mKO the same as Kate? I afraid that I don't have any experience with mKO,
> but do know that you will not be able to image mCherry with your laser.
> mCherry requires wavelengths longer than 1,000 nm.
> I do have a dsRed variant to suggest though! The dsRed express variant sold
> by Clontech is very bright and easy to excite with both a two-photon laser
> and the 568 laser. It is compatible with excitation of GFP and CFP at a
> wavelength of 865 nm with the 2p laser. I would definitely suggest that
> they buy this fluorophore instead!
> There is a strain of mice that expresses this ubiquitously sold by Jax made
> by the Nagy lab. I use it all the time and love it. The fluorescent protein
> itself was engineered by the Glick lab (chicago I think)
> best,
> Ann
>
> I have a researcher trying to plan future experiments with fluorescent
> proteins and we are trying to determine what the best options would be based
> on our equipment set. They are considering mCherry vs mKO and are leaning
> toward mKO (I think there is an mKO2 now) because the literature shows it to
> be brighter and more stable. They would like to do in vivo work with 2
> photon (we have a Prairie Ultima 2 with a Coherent Chameleon XR laser: 720 -
> 980nm). Ideally, they would also like to do single photon confocal, but we
> are limited to a 568 laser at the moment (hoping to upgrade soon), which
> seems to be just a bit too high for mKO.
>
> Has anyone worked with these proteins (especially in vivo) who could give me
> an idea of the excitation peaks with standard confocal and 2-P?
> Characterization of mCherry is pretty good, but there is very little to go
> on with the mKO. Any help pointing to some literature would be most
> appreciated as well.
>
> James E. Hayden, RBP, FBCA
>
> Manager
>
> Microscopy Core Facility
>
> The Wistar Institute
>
> 3601 Spruce St.
>
> Philadelphia, PA 19104
>
> (215)898-3887
>
> [hidden email]
>
> --
>
> Ann Haberman, PhD
> Department of Laboratory Medicine
> Yale University School of Medicine
> 1 Gilbert St.
> TAC S541
> New Haven, CT 06510
>
> 203-785-7349
> 203-785-5415 (fax)
> [hidden email]
>

Vitaly Boyko

Re: mKO with 2-Photon

In reply to this post by Jamie Hayden

Hi James,

I have compared mKO with mCherry, mStrawberry and other fluor. proteins on the left as a fusions to a non-monomeric, often membrane associated proteins. Even CD8-mKO fusion (a dimer) yielded highly negative results (high cytotoxicity, aggregation on membranes etc.). mCherry was not as good as venusYFP or EYFP, but rather close to YFPs than to mKO. There were minor differences between mStraberry and mCherry, with mCherry being slightly better in revealing its stickiness to membranes, as mCherry seems not to be monomeric as reported (has been confirmed by the YFP based BiFC assay).That might explain its visible membrane cytotoxicity. But you can live with it, but not with mKO based fusions.

Good luck,

Vitaly

From: Jamie Hayden <[hidden email]>
To: [hidden email]
Sent: Thu, November 12, 2009 11:19:19 AM
Subject: mKO with 2-Photon

I have a researcher trying to plan future experiments with fluorescent proteins and we are trying to determine what the best options would be based on our equipment set. They are considering mCherry vs mKO and are leaning toward mKO (I think there is an mKO2 now) because the literature shows it to be brighter and more stable. They would like to do in vivo work with 2 photon (we have a Prairie Ultima 2 with a Coherent Chameleon XR laser: 720 - 980nm). Ideally, they would also like to do single photon confocal, but we are limited to a 568 laser at the moment (hoping to upgrade soon), which seems to be just a bit too high for mKO.

James E. Hayden, RBP, FBCA

Manager

Microscopy Core Facility

The Wistar Institute

3601 Spruce St.

Philadelphia, PA 19104

(215)898-3887

[hidden email]

Adrian Smith-6

Re: mKO with 2-Photon

In reply to this post by Haberman, Ann

Hi all,

I think mKO is Kusabira Orange.

Finding a good single photon spectra is hard enough let alone a 2P.

The best I've found is the MBL product literature and the Cell paper which described the FUCCI construct with the original description (I think) of mKO2 (don't think it has spectra).

http://www.mblintl.com/sites/default/files/MBLI%20FUCCI%20Flyer.pdf

http://www.cell.com/abstract/S0092-8674(08)00054-8

I have users who have looked at FUCCI contructs from MBL and the mKO2 is fine with a 561nm line on our SP5 - sorry haven't got 568nm line to test. We have been able to analyse it on flow with a 488nm line.

Sorry, no detailed 2P work yet but my impression is that you will get better excitation from your laser for mKO2 than you will for mCherry.

Interestingly I just noticed that Invitrogen are selling a BacMan kit with the FUCCI sensor where they talk about "GFP" and "RFP" - not clear if they are just using generic terms or they have actually changed the proteins (I would doubt it).

http://products.invitrogen.com/ivgn/product/P36232?CID=fl-fucci

Regards,

Adrian

On 13/11/2009, at 5:22 AM, Ann Haberman wrote:

Hi Jamie,

Is mKO the same as Kate? I afraid that I don't have any experience with mKO, but do know that you will not be able to image mCherry with your laser. mCherry requires wavelengths longer than 1,000 nm.

I do have a dsRed variant to suggest though! The dsRed express variant sold by Clontech is very bright and easy to excite with both a two-photon laser and the 568 laser. It is compatible with excitation of GFP and CFP at a wavelength of 865 nm with the 2p laser. I would definitely suggest that they buy this fluorophore instead!

There is a strain of mice that expresses this ubiquitously sold by Jax made by the Nagy lab. I use it all the time and love it. The fluorescent protein itself was engineered by the Glick lab (chicago I think)

best,
Ann

I have a researcher trying to plan future experiments with fluorescent proteins and we are trying to determine what the best options would be based on our equipment set. They are considering mCherry vs mKO and are leaning toward mKO (I think there is an mKO2 now) because the literature shows it to be brighter and more stable. They would like to do in vivo work with 2 photon (we have a Prairie Ultima 2 with a Coherent Chameleon XR laser: 720 - 980nm). Ideally, they would also like to do single photon confocal, but we are limited to a 568 laser at the moment (hoping to upgrade soon), which seems to be just a bit too high for mKO.

Has anyone worked with these proteins (especially in vivo) who could give me an idea of the excitation peaks with standard confocal and 2-P? Characterization of mCherry is pretty good, but there is very little to go on with the mKO. Any help pointing to some literature would be most appreciated as well.
James E. Hayden, RBP, FBCA
Manager
Microscopy Core Facility
The Wistar Institute
3601 Spruce St.
Philadelphia, PA 19104

(215)898-3887
[hidden email]

--

Ann Haberman, PhD
Department of Laboratory Medicine
Yale University School of Medicine
1 Gilbert St.
TAC S541
New Haven, CT 06510

203-785-7349
203-785-5415 (fax)
[hidden email]

George McNamara

Re: mKO with 2-Photon

In reply to this post by Jamie Hayden

Hi Jamie,

Kremers et al 2009 Nat Methods reported a small "photoconversion" wavelength shift of mKO and cited a paper on photoconversion of mKO with 436 nm excitation to a green fluorescent form (their ref 14: Goedhart, J., Vermeer, J.E., Adjobo-Hermans, M.J., van Weeren, L. & Gadella, T.W. Jr. PLoS ONE 2 , e1011 (2007).). Kremers et al reported that many "orange" fluorescent proteins have some kind of photoconversion. A small spectral shift may not be a problem if the cells being studied will only have one FP, but users try to multiplex FPs, spectral shifting should raise the bar on appropriate controls.

In Kremers hands, mCherry never exhibited any type of photoconversion.

Has anyone seen mKate having blue excitation -> green emission on "first light" (first illumination of a field of view)? Kremers reported photoconversion of mKate to green emission but a colleague here has found that mKate expressing cells have noticeable green emission at "first light (with the same parental cell line, their equivalent mCherry-histone H2B never has green emission).

My understanding of mCherry is that closer to 3p excitation may work better than theoretical "divide excitation wavelength peak by 2". This might have as much to do with most MP lasers having more power in the 720-850 nm range than above 950 nm, and/or optics transmission efficiency, but suggests that (1) empirical wavelength tuning should be performed for each FP and imaging rig and (2) the term "multiphoton excitation" should be used unless authors have a clear best fit of 2photon laser power vs fluorescence emission.

George

At 11:19 AM 11/12/2009, you wrote:

I have a researcher trying to plan future experiments with fluorescent proteins and we are trying to determine what the best options would be based on our equipment set. They are considering mCherry vs mKO and are leaning toward mKO (I think there is an mKO2 now) because the literature shows it to be brighter and more stable. They would like to do in vivo work with 2 photon (we have a Prairie Ultima 2 with a Coherent Chameleon XR laser: 720 - 980nm). Ideally, they would also like to do single photon confocal, but we are limited to a 568 laser at the moment (hoping to upgrade soon), which seems to be just a bit too high for mKO.

Has anyone worked with these proteins (especially in vivo) who could give me an idea of the excitation peaks with standard confocal and 2-P? Characterization of mCherry is pretty good, but there is very little to go on with the mKO. Any help pointing to some literature would be most appreciated as well.

James E. Hayden, RBP, FBCA
Manager
Microscopy Core Facility
The Wistar Institute
3601 Spruce St.
Philadelphia, PA 19104

(215)898-3887
[hidden email]

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

Periasamy, Ammasi (ap3t)

Controversial FRET results...........

Dear Friends

I just wanted to inform you that Dr. Steven Vogel, NIH, just published a paper on his studies of FRET with multiple FP acceptors in PLoS ONE. The manuscript can be accessed at:

http://dx.plos.org/10.1371/journal.pone.0008031

As the results Dr. Vogel present in this manuscript have been controversial when he presented them at several meetings, posters, and with reviewers, would like to inform you about a unique feature of PLoS ONE, readers can post comment, blog, etc. on the published manuscripts by clicking on the metrics tab on the top of the article web page (directly below the title). It is Dr. Vogel’s hope, and one of the reasons he decided to publish this study in PLoS ONE, that he can generate a scientific dialog on this aspect of FRET, and on his curious and mysterious observations.

Happy Holidays!

Ammasi Periasamy, Ph.D.

Director, Keck Center for Cellular Imaging (KCCI)

Professor of Biology and Biomedical Engineering

Biology, Gilmer Hall (064), McCormick Rd

University of Virginia

Charlottesville, VA 22904

Voice: 434-243-7602 (Office); 982-4869 (lab)

Fax:434-982-5210; Email:[hidden email]

http://www.kcci.virginia.edu

************************

Workshop on FRET Microscopy, March 9-13, 2010

http://www.kcci.virginia.edu/workshop/workshop2010/index.php

*************************

John Oreopoulos

Re: Controversial FRET results...........

Dr. Periasamy,

Could you explain in further detail the reasons for why this work was considered controversial at the time? Is it simply because no explanation of what this mysterious additional energy transfer pathway is was given? What are you thoughts on the interpretation of the data presented? How does the conclusion affect the average person doing a FRET imaging experiment (if at all)?

John Oreopoulos

On 30-Nov-09, at 11:06 AM, Periasamy, Ammasi (ap3t) wrote:

Dear Friends
I just wanted to inform you that Dr. Steven Vogel, NIH, just published a paper on his studies of FRET with multiple FP acceptors in PLoS ONE. The manuscript can be accessed at:

http://dx.plos.org/10.1371/journal.pone.0008031

As the results Dr. Vogel present in this manuscript have been controversial when he presented them at several meetings, posters, and with reviewers, would like to inform you about a unique feature of PLoS ONE, readers can post comment, blog, etc. on the published manuscripts by clicking on the metrics tab on the top of the article web page (directly below the title). It is Dr. Vogel’s hope, and one of the reasons he decided to publish this study in PLoS ONE, that he can generate a scientific dialog on this aspect of FRET, and on his curious and mysterious observations.

Happy Holidays!

Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI)
Professor of Biology and Biomedical Engineering
Biology, Gilmer Hall (064), McCormick Rd
University of Virginia
Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:434-982-5210; Email:[hidden email]
http://www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 9-13, 2010
http://www.kcci.virginia.edu/workshop/workshop2010/index.php
*************************

Vitaly Boyko

Re: Controversial FRET results...........

Dear John,

It might be more appropriate if we can discuss the topic through the PlosOne, so that Dr. Vogel would be available for a comment.

However, live cell FRET is a controversial system on its own, as particular donor-acceptor constructs could be overexpressed to different levels (1 ug of plasmid per 35 mm dish, ca. 600'000 cells, is quite a bit for the 293 or 293T cells), or reveal different "stickiness" to membranes. As far as there is no easy way to monitor donor-acceptor concentration on a voxel-by-voxel basis over time, the FRET data could be easily, often unintentionally, "manipulated". Moreover, there is no easy control experiment for the possible and likely aggregation state of the donor-acceptor molecules/chimeras (as wt and the amber mutant could have different aggregation capabilities). It is very serious and fundamental issue, especially when proteins of interest and their mutants have been analyzed. Different mutants or differently tagged proteins are often mislocalized (plenty of examples with the short FLAG-, HA- or myc-tagged proteins). The same is, unfortunately, also applicable to the FLIM FRET.

The FRET experiments are very difficult to perform highly accurately, especially under physiological conditions. Dave Piston's CeFP is a good donor (also carrying the A206K monomeric mutation - but I have some doubts that a single mutation can keep it monomeric at the Kd of 74 mM, calculated by extrapolation in vitro - correct me John, if I am wrong). But in general CeFP channel is quite noisy, both optically and biologically.

Well, we may need to wait till, for example FCS, FLIM and Spinning Disk would get functionally integrated to reach a capability to work at physiological concentrations of biomolecules.

Let's be patient and let's hope we get a 8 um/pixel EM-CCD cameras, which can outperform the Evolve, ImageM or iXon EM-CCDs (very good cameras though) - it may take a decade or so, that's life!

Cheers,

Vitaly

301-515-7833

From: John Oreopoulos <[hidden email]>
To: [hidden email]
Sent: Mon, November 30, 2009 3:20:02 PM
Subject: Re: Controversial FRET results...........

Dr. Periasamy,

John Oreopoulos

On 30-Nov-09, at 11:06 AM, Periasamy, Ammasi (ap3t) wrote:

Dear Friends

I just wanted to inform you that Dr. Steven Vogel, NIH, just published a paper on his studies of FRET with multiple FP acceptors in PLoS ONE. The manuscript can be accessed at:

http://dx.plos.org/10.1371/journal.pone.0008031

As the results Dr. Vogel present in this manuscript have been controversial when he presented them at several meetings, posters, and with reviewers, would like to inform you about a unique feature of PLoS ONE, readers can post comment, blog, etc. on the published manuscripts by clicking on the metrics tab on the top of the article web page (directly below the title). It is Dr. Vogel’s hope, and one of the reasons he decided to publish this study in PLoS ONE, that he can generate a scientific dialog on this aspect of FRET, and on his curious and mysterious observations.

Happy Holidays!

Ammasi Periasamy, Ph.D.

Director, Keck Center for Cellular Imaging (KCCI)

Professor of Biology and Biomedical Engineering

Biology, Gilmer Hall (064), McCormick Rd

University of Virginia

Charlottesville, VA 22904

Voice: 434-243-7602 (Office); 982-4869 (lab)

Fax:434-982-5210; Email:[hidden email]

http://www.kcci.virginia.edu

************************

Workshop on FRET Microscopy, March 9-13, 2010

http://www.kcci.virginia.edu/workshop/workshop2010/index.php

*************************

Periasamy, Ammasi (ap3t)

Re: Controversial FRET results...........

In reply to this post by John Oreopoulos

Sorry, I was in my class.

Please post your questions in Plos One so that Steven can answer directly post the answer in PLos One. I also forwarded your questions to him.

Best,

ammasi

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Monday, November 30, 2009 3:20 PM
To: [hidden email]
Subject: Re: Controversial FRET results...........

Dr. Periasamy,

John Oreopoulos

On 30-Nov-09, at 11:06 AM, Periasamy, Ammasi (ap3t) wrote:

Dear Friends

I just wanted to inform you that Dr. Steven Vogel, NIH, just published a paper on his studies of FRET with multiple FP acceptors in PLoS ONE. The manuscript can be accessed at:

http://dx.plos.org/10.1371/journal.pone.0008031

Happy Holidays!

Ammasi Periasamy, Ph.D.

Director, Keck Center for Cellular Imaging (KCCI)

Professor of Biology and Biomedical Engineering

Biology, Gilmer Hall (064), McCormick Rd

University of Virginia

Charlottesville, VA 22904

Voice: 434-243-7602 (Office); 982-4869 (lab)

Fax:434-982-5210; Email:[hidden email]

http://www.kcci.virginia.edu

************************

Workshop on FRET Microscopy, March 9-13, 2010

http://www.kcci.virginia.edu/workshop/workshop2010/index.php

*************************

Emmanuel Gustin

Re: Controversial FRET results...........

In reply to this post by Periasamy, Ammasi (ap3t)

Hello,

Maybe I am missing something, but why is it surprising that the simple(st) kinetic model does not provide a fully accurate prediction in a case like this?

The assumption behind that approximation seems to be that the donor is in close enough proximity with the acceptors to have FRET, but that the acceptors are sufficiently separated not to have any coupling behind their electronic states or more indirect interaction... At first sight, that is not a very likely assumption. And any coupling between the acceptors would result in an additional term to their potential energy functions and therefore small shifts in their electronic energy states. Which is always likely to have at least some effect on their fluorescence efficiency and the FRET efficiency with the donor.

It’s a pity that they didn’t measure fluorescence lifetime for the acceptor molecules in different constructs, as this would have given at least a hint about the possible influence of acceptor-acceptor coupling.

Best Regards,

Emmanuel

--
Emmanuel Gustin, Tel. (+32) 15 46 1586, e-mail: [hidden email]

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t)
Sent: maandag 30 november 2009 17:07
To: [hidden email]
Subject: Controversial FRET results...........

Dear Friends

I just wanted to inform you that Dr. Steven Vogel, NIH, just published a paper on his studies of FRET with multiple FP acceptors in PLoS ONE. The manuscript can be accessed at:

http://dx.plos.org/10.1371/journal.pone.0008031

Happy Holidays!

Ammasi Periasamy, Ph.D.

Director, Keck Center for Cellular Imaging (KCCI)

Professor of Biology and Biomedical Engineering

Biology, Gilmer Hall (064), McCormick Rd

University of Virginia

Charlottesville, VA 22904

Voice: 434-243-7602 (Office); 982-4869 (lab)

Fax:434-982-5210; Email:[hidden email]

http://www.kcci.virginia.edu

************************

Workshop on FRET Microscopy, March 9-13, 2010

http://www.kcci.virginia.edu/workshop/workshop2010/index.php

*************************

Mark Cannell

Re: Controversial FRET results...........

Hi Emmanuel

This would upset the common interpretation of FRET changes only in terms
of a change in distance or in a highly specific interaction between a
single acceptor and donor.

I think that the paper could have come out and said that it is most
likely that the interactions are not independent. consideration of FRET
as one form of polariton decay should cause one to realize that you
can't consider FRET changes solely in terms of distance. If the crowding
of other proteins changes the local resonance environment (as required
for any FRET to occur in the first place) FRET efficiency/life time
might well change as the probability of polariton decay via emission of
a longer wavelength photon is changed.

What the paper shows is that the process is not linear -but I think
there is precident for that: the rate constant of a chemical reaction
not simply determined by RT but is also determined by the number of
vibrational modes available that can temporarily contribute RTs to the
creation of the activated state. This introduces some higher power terms
so that the overall reaction rate is not proprotional to the number of
modes present.

As I said in an earlier post, I continue to be bemused by the simplistic
interpretation of FRET changes only in terms of distance...

Regards Mark Cannell

>
> Maybe I am missing something, but why is it surprising that the
> simple(st) kinetic model does not provide a fully accurate prediction
> in a case like this?
>
> The assumption behind that approximation seems to be that the donor is
> in close enough proximity with the acceptors to have FRET, but that
> the acceptors are sufficiently separated not to have any coupling
> behind their electronic states or more indirect interaction... At
> first sight, that is not a very likely assumption. And any coupling
> between the acceptors would result in an additional term to their
> potential energy functions and therefore small shifts in their
> electronic energy states. Which is always likely to have at least some
> effect on their fluorescence efficiency and the FRET efficiency with
> the donor.
>
> It’s a pity that they didn’t measure fluorescence lifetime for the
> acceptor molecules in different constructs, as this would have given
> at least a hint about the possible influence of acceptor-acceptor
> coupling.
>
> Best Regards,
>
> Emmanuel
>
> --
> Emmanuel Gustin, Tel. (+32) 15 46 1586, e-mail: [hidden email]
> <mailto:[hidden email]>
>
> *From:* Confocal Microscopy List
> [mailto:[hidden email]] *On Behalf Of *Periasamy,
> Ammasi (ap3t)
> *Sent:* maandag 30 november 2009 17:07
> *To:* [hidden email]
> *Subject:* Controversial FRET results...........
>
> Dear Friends
>
> I just wanted to inform you that Dr. Steven Vogel, NIH, just published
> a paper on his studies of FRET with multiple FP acceptors in PLoS ONE.
> The manuscript can be accessed at:
>
> http://dx.plos.org/10.1371/journal.pone.0008031
>
> As the results Dr. Vogel present in this manuscript have been
> controversial when he presented them at several meetings, posters, and
> with reviewers, would like to inform you about a unique feature of
> PLoS ONE, readers can post comment, blog, etc. on the published
> manuscripts by clicking on the metrics tab on the top of the article
> web page (directly below the title). It is Dr. Vogel’s hope, and one
> of the reasons he decided to publish this study in PLoS ONE, that he
> can generate a scientific dialog on this aspect of FRET, and on his
> curious and mysterious observations.
>
> Happy Holidays!
>
> Ammasi Periasamy, Ph.D.
>
> Director, Keck Center for Cellular Imaging (KCCI)
>
> Professor of Biology and Biomedical Engineering
>
> Biology, Gilmer Hall (064), McCormick Rd
>
> University of Virginia
>
> Charlottesville, VA 22904
>
> Voice: 434-243-7602 (Office); 982-4869 (lab)
>
> Fax:434-982-5210; Email:[hidden email]
>
> http://www.kcci.virginia.edu
>
> ************************
>
> Workshop on FRET Microscopy, March 9-13, 2010
>
> http://www.kcci.virginia.edu/workshop/workshop2010/index.php
>
> *************************
>