Laser effects during bleaching

[John Runions]

Hi Tom,

When doing FRAP, we generally try to keep the bleaching lasers as low as possible so that a loss of fluorescence results but so that we aren't applying enough laser power to damage other proteins in membranes.  If the lasers were so hot as to denature proteins the whole procedure would be invalidated as we'd be killing the cells (I do often imagine the sound of frying bacon during the bleaching phase and hope that it's not really happening!).

At normal laser power levels for confocal, cells seem to function properly over the relatively long term.  The trick during the bleaching phase is to increase the laser power so that a bit of bleaching results.  If the region to be studied is bleached completely black, then I feel there really is the potential for cellular damage.  I've certainly exploded cells with too much laser power so your specimens should be handled gently.  The small amount of bleaching attained using reasonable laser power is analyzed relativistically, i.e. you normalize post-bleach and pre-bleach intensity between 0-100% intensity so that there is no requirement to bleach the fluorescence completely away.  The idea of fluorescence recovery is that any molecule will move to its equilibrium concentration in time (unless constrained).  The species of molecule that we are analyzing the dynamics of in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP can be done, therefore, with a small population of bleached molecules.

In some recent experiments, we have been FRAPing so that only a very small decrease in fluorescence occurs within a region of interest.  Recovery curves fit from these experiments seem equivalent to those fit to data sets in which much more bleaching of the ROI was visible.  We need to ascertain if they are significantly the same, however.  If any physicists of diffusion are listening it would be nice to have your take on this.

Regards, John.

Donnelly, Tom wrote:

Just to add to the confusion.

 

What happens to the non-fluorescent proteins in the cell when you crank up the power to bleach or image in live cells?

 

Tom

-----Original Message-----
From: Confocal Microscopy List [[hidden email]]On Behalf Of John Runions
Sent: Tuesday, November 18, 2008 9:08 AM
To: [hidden email]
Subject: Re: broadband excitation vs. narrow band

Boy, what a stupid question Carl.  I think we should all fail to dignify it with a response!

Actually, that kind of question is fundamental to us in the FRAP world and it sounds like someone needs to do some good empirical measuring.  When bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the argon laser at the same time.  It works better that just using the 488 and I explain this by saying that it is because we are bleaching with a broader spectrum across the excitation range.  I have never been able (or tried) to confirm if this is the case or if the higher power at the specimen plays a role.

Sorry to be not much help.  John.

Carl Boswell wrote:

Hi all,
I was told once that there are no stupid questions, so let's test that assumption.

The question has to do with photobleaching vs. excitation energy.  To get X photons from a fluor, would there be less photobleaching using a single wavelength excitation source at peak excitation wavelength, or a broadband (20-30nm) light source centered on the peak excitation of the molecule, or would there be no difference?  My assumption is that lower "power" (brightness?) would be needed for the broadband source, but would the overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from "inefficient" excitation, i.e. off-peak excitation, to get the same output? If a fluor is less efficiently excited, is it less efficiently bleached, even though more power may be needed to get equivalent output?

Thanks,
Carl

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

[Jonathan M Krupp]

Just a quick note to thank everyone for your ideas about
finding simple demos for fluorescence imaging. I have
tried several with great results.

So, now that I have that under control, here is another
question.

I am part of  an EM training program, to which I would
like to add more light microscopy. We have a moderate
range of LM instrumentation, but not a confocal scope.
What sorts of things would be useful to add to our program
to prepare technical level students to become contributing
members of microscopy labs? Brainstorming is encouraged.
What do you look for when adding technical help to your
operation?

If I could do it, I would add a confocal, but $$ is a
problem. Any suggestions on where to find used scopes
and/or what features/models would work in an introductory
program?

Thanks again, you are all a great help to me.

Jon

[John Runions]

Hi again Tom, I also meant to say in the last email that I sent you that bleaching of a fluorescent molecule should result because it loses electrons irreversibly during repeated rounds of excitation and emission - not because it is damaged or it's structure is altered in any way by the laser power used.  If that is the case, then there shouldn't be any damage to any other proteins either.  This is a difficult proposition to prove so we usually operate on the assumption that if the membrane or organelle being bleached recovers and the cell continues top live happily then the laser power was below damaging levels.  John.

Donnelly, Tom wrote:

Just to add to the confusion.

 

What happens to the non-fluorescent proteins in the cell when you crank up the power to bleach or image in live cells?

 

Tom

-----Original Message-----
From: Confocal Microscopy List [[hidden email]]On Behalf Of John Runions
Sent: Tuesday, November 18, 2008 9:08 AM
To: [hidden email]
Subject: Re: broadband excitation vs. narrow band

Boy, what a stupid question Carl.  I think we should all fail to dignify it with a response!

Actually, that kind of question is fundamental to us in the FRAP world and it sounds like someone needs to do some good empirical measuring.  When bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the argon laser at the same time.  It works better that just using the 488 and I explain this by saying that it is because we are bleaching with a broader spectrum across the excitation range.  I have never been able (or tried) to confirm if this is the case or if the higher power at the specimen plays a role.

Sorry to be not much help.  John.

Carl Boswell wrote:

Hi all,
I was told once that there are no stupid questions, so let's test that assumption.

The question has to do with photobleaching vs. excitation energy.  To get X photons from a fluor, would there be less photobleaching using a single wavelength excitation source at peak excitation wavelength, or a broadband (20-30nm) light source centered on the peak excitation of the molecule, or would there be no difference?  My assumption is that lower "power" (brightness?) would be needed for the broadband source, but would the overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from "inefficient" excitation, i.e. off-peak excitation, to get the same output? If a fluor is less efficiently excited, is it less efficiently bleached, even though more power may be needed to get equivalent output?

Thanks,
Carl

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

[Kevin Braeckmans]

Hi,

 

In fact, generally speaking, this is not the case. For example, the recovery half-time generally depends on the amount of photobleaching. There is one exception: the photobleaching of a 2-D uniform disk. This is clearly explained in the ‘father of all FRAP articles’: Axelrod et al 1976, Biophys J 16.

 

For the same reason I strongly discourage the normalization to the post-photobleaching level (i.e. putting the post-photobleaching level to 0%)! If you want to compare FRAP curves, they all should have been acquired using exactly the same settings, including the amount of photobleaching. Therefore, there is no need at all to do the post-photobleaching normalization, except to mask the differences between different experiments… I know that in many semi-quantitative FRAP articles this is being done, but I would not recommend this since it is physically incorrect. That being said, for small amounts of photobleaching, small differences do not matter so much (see e.g. fig 7 of the Axelrod paper in case of a 2-D spot photobleached by a stationary Gaussian beam).

 

Hope this helps,

 

Best regards,

 

Kevin

 

 

 

Prof. Dr. Kevin Braeckmans

Lab. General Biochemistry and Physical Pharmacy

Ghent University

Harelbekestraat 72

9000 Ghent

Belgium

Tel: [phone]

Fax: [phone]

 

 

 

 

 

 

 

Van: Confocal Microscopy List [mailto:[hidden email]] Namens John Runions
Verzonden: dinsdag 18 november 2008 18:37
Aan: [hidden email]
Onderwerp: Laser effects during bleaching

 

Hi Tom,

When doing FRAP, we generally try to keep the bleaching lasers as low as possible so that a loss of fluorescence results but so that we aren't applying enough laser power to damage other proteins in membranes.  If the lasers were so hot as to denature proteins the whole procedure would be invalidated as we'd be killing the cells (I do often imagine the sound of frying bacon during the bleaching phase and hope that it's not really happening!).

At normal laser power levels for confocal, cells seem to function properly over the relatively long term.  The trick during the bleaching phase is to increase the laser power so that a bit of bleaching results.  If the region to be studied is bleached completely black, then I feel there really is the potential for cellular damage.  I've certainly exploded cells with too much laser power so your specimens should be handled gently.  The small amount of bleaching attained using reasonable laser power is analyzed relativistically, i.e. you normalize post-bleach and pre-bleach intensity between 0-100% intensity so that there is no requirement to bleach the fluorescence completely away.  The idea of fluorescence recovery is that any molecule will move to its equilibrium concentration in time (unless constrained).  The species of molecule that we are analyzing the dynamics of in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP can be done, therefore, with a small population of bleached molecules.

In some recent experiments, we have been FRAPing so that only a very small decrease in fluorescence occurs within a region of interest.  Recovery curves fit from these experiments seem equivalent to those fit to data sets in which much more bleaching of the ROI was visible.  We need to ascertain if they are significantly the same, however.  If any physicists of diffusion are listening it would be nice to have your take on this.

Regards, John.

Donnelly, Tom wrote:

Just to add to the confusion.

 

What happens to the non-fluorescent proteins in the cell when you crank up the power to bleach or image in live cells?

 

Tom

-----Original Message-----
From: Confocal Microscopy List [[hidden email]]On Behalf Of John Runions
Sent: Tuesday, November 18, 2008 9:08 AM
To: [hidden email]
Subject: Re: broadband excitation vs. narrow band

Boy, what a stupid question Carl.  I think we should all fail to dignify it with a response!

Actually, that kind of question is fundamental to us in the FRAP world and it sounds like someone needs to do some good empirical measuring.  When bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the argon laser at the same time.  It works better that just using the 488 and I explain this by saying that it is because we are bleaching with a broader spectrum across the excitation range.  I have never been able (or tried) to confirm if this is the case or if the higher power at the specimen plays a role.

Sorry to be not much help.  John.

Carl Boswell wrote:

Hi all,
I was told once that there are no stupid questions, so let's test that assumption.

The question has to do with photobleaching vs. excitation energy.  To get X photons from a fluor, would there be less photobleaching using a single wavelength excitation source at peak excitation wavelength, or a broadband (20-30nm) light source centered on the peak excitation of the molecule, or would there be no difference?  My assumption is that lower "power" (brightness?) would be needed for the broadband source, but would the overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from "inefficient" excitation, i.e. off-peak excitation, to get the same output? If a fluor is less efficiently excited, is it less efficiently bleached, even though more power may be needed to get equivalent output?

Thanks,
Carl

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

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

 

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

[John Chandler-2]

So, Jon,

Which demo samples do you like?  Which ones have good effort to results ratio?

Inquiring minds want to know.

--John

>===== Original Message From Confocal Microscopy List
<[hidden email]> =====
>Just a quick note to thank everyone for your ideas about
>finding simple demos for fluorescence imaging. I have
>tried several with great results.
>
>So, now that I have that under control, here is another
>question.
>

[John Runions]

Whew, I'm glad you jumped in there Kevin.  The Axelrod paper is the paper that explains things but I find the math very dense.  Within a given experiment, we bleach a 2D uniform disk and I do try to control so that the level of photobleaching is approximately the same each time.  But, I don't necessarily  control between experiments and I have been happily letting people compare results from different experiments that used different bleaching conditions and levels.

The type of FRAP we are doing is only semi-quantitative.  In fact, I have been calling the half-time of recovery the 'relative diffusion coefficient'. This is because there are too many unknowns in the cell membranes we study to derive the actual diffusion coefficient.

From now on, I will stress the importance of bleaching to the same approximate level each time.

Thanks for your help.  John.

Kevin Braeckmans wrote:

Hi,

 

In fact, generally speaking, this is not the case. For example, the recovery half-time generally depends on the amount of photobleaching. There is one exception: the photobleaching of a 2-D uniform disk. This is clearly explained in the ‘father of all FRAP articles’: Axelrod et al 1976, Biophys J 16.

 

For the same reason I strongly discourage the normalization to the post-photobleaching level (i.e. putting the post-photobleaching level to 0%)! If you want to compare FRAP curves, they all should have been acquired using exactly the same settings, including the amount of photobleaching. Therefore, there is no need at all to do the post-photobleaching normalization, except to mask the differences between different experiments… I know that in many semi-quantitative FRAP articles this is being done, but I would not recommend this since it is physically incorrect. That being said, for small amounts of photobleaching, small differences do not matter so much (see e.g. fig 7 of the Axelrod paper in case of a 2-D spot photobleached by a stationary Gaussian beam).

 

Hope this helps,

 

Best regards,

 

Kevin

 

 

 

Prof. Dr. Kevin Braeckmans

Lab. General Biochemistry and Physical Pharmacy

Ghent University

Harelbekestraat 72

9000 Ghent

Belgium

Tel: [phone]

Fax: [phone]

 

 

 

 

 

 

 

Van: Confocal Microscopy List [[hidden email]] Namens John Runions
Verzonden: dinsdag 18 november 2008 18:37
Aan: [hidden email]
Onderwerp: Laser effects during bleaching

 

Hi Tom,

When doing FRAP, we generally try to keep the bleaching lasers as low as possible so that a loss of fluorescence results but so that we aren't applying enough laser power to damage other proteins in membranes.  If the lasers were so hot as to denature proteins the whole procedure would be invalidated as we'd be killing the cells (I do often imagine the sound of frying bacon during the bleaching phase and hope that it's not really happening!).

At normal laser power levels for confocal, cells seem to function properly over the relatively long term.  The trick during the bleaching phase is to increase the laser power so that a bit of bleaching results.  If the region to be studied is bleached completely black, then I feel there really is the potential for cellular damage.  I've certainly exploded cells with too much laser power so your specimens should be handled gently.  The small amount of bleaching attained using reasonable laser power is analyzed relativistically, i.e. you normalize post-bleach and pre-bleach intensity between 0-100% intensity so that there is no requirement to bleach the fluorescence completely away.  The idea of fluorescence recovery is that any molecule will move to its equilibrium concentration in time (unless constrained).  The species of molecule that we are analyzing the dynamics of in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP can be done, therefore, with a small population of bleached molecules.

In some recent experiments, we have been FRAPing so that only a very small decrease in fluorescence occurs within a region of interest.  Recovery curves fit from these experiments seem equivalent to those fit to data sets in which much more bleaching of the ROI was visible.  We need to ascertain if they are significantly the same, however.  If any physicists of diffusion are listening it would be nice to have your take on this.

Regards, John.

Donnelly, Tom wrote:

Just to add to the confusion.

 

What happens to the non-fluorescent proteins in the cell when you crank up the power to bleach or image in live cells?

 

Tom

-----Original Message-----
From: Confocal Microscopy List [[hidden email]]On Behalf Of John Runions
Sent: Tuesday, November 18, 2008 9:08 AM
To: [hidden email]
Subject: Re: broadband excitation vs. narrow band

Boy, what a stupid question Carl.  I think we should all fail to dignify it with a response!

Actually, that kind of question is fundamental to us in the FRAP world and it sounds like someone needs to do some good empirical measuring.  When bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the argon laser at the same time.  It works better that just using the 488 and I explain this by saying that it is because we are bleaching with a broader spectrum across the excitation range.  I have never been able (or tried) to confirm if this is the case or if the higher power at the specimen plays a role.

Sorry to be not much help.  John.

Carl Boswell wrote:

Hi all,
I was told once that there are no stupid questions, so let's test that assumption.

The question has to do with photobleaching vs. excitation energy.  To get X photons from a fluor, would there be less photobleaching using a single wavelength excitation source at peak excitation wavelength, or a broadband (20-30nm) light source centered on the peak excitation of the molecule, or would there be no difference?  My assumption is that lower "power" (brightness?) would be needed for the broadband source, but would the overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from "inefficient" excitation, i.e. off-peak excitation, to get the same output? If a fluor is less efficiently excited, is it less efficiently bleached, even though more power may be needed to get equivalent output?

Thanks,
Carl

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

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

 

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

[Kevin Braeckmans]

Hi John,

 

 

Yes, I appreciate that the maths can be challenging, however they tell us what can and cannot be done. In this particular case you could skip the maths and have a look at the experimental section, this is where it is actually explained without too much maths.

 

Just to make sure, if you want to compare recovery half times between experiments, not only the amount of photobleaching but the entire experimental set-up has to be the same: same size of photobleaching disk, same objective lens, same pinhole setting, same space around the photobleaching area – they all enter the equation. Some argue that this is not so important if you are just interested in a rough measurement. The thing is that with so many unknowns, you don’t have a clue anymore of the accuracy of the values that are obtained.

 

Sorry, don’t want to sound too strict, this is just my take on the subject.

 

Good luck and best regards,

 

Kevin

 

 

 

 

 

Van: Confocal Microscopy List [mailto:[hidden email]] Namens John Runions
Verzonden: woensdag 19 november 2008 11:21
Aan: [hidden email]
Onderwerp: Re: Laser effects during bleaching

 

Whew, I'm glad you jumped in there Kevin.  The Axelrod paper is the paper that explains things but I find the math very dense.  Within a given experiment, we bleach a 2D uniform disk and I do try to control so that the level of photobleaching is approximately the same each time.  But, I don't necessarily  control between experiments and I have been happily letting people compare results from different experiments that used different bleaching conditions and levels.

The type of FRAP we are doing is only semi-quantitative.  In fact, I have been calling the half-time of recovery the 'relative diffusion coefficient'. This is because there are too many unknowns in the cell membranes we study to derive the actual diffusion coefficient.

From now on, I will stress the importance of bleaching to the same approximate level each time.

Thanks for your help.  John.

Kevin Braeckmans wrote:

Hi,

 

In fact, generally speaking, this is not the case. For example, the recovery half-time generally depends on the amount of photobleaching. There is one exception: the photobleaching of a 2-D uniform disk. This is clearly explained in the ‘father of all FRAP articles’: Axelrod et al 1976, Biophys J 16.

 

For the same reason I strongly discourage the normalization to the post-photobleaching level (i.e. putting the post-photobleaching level to 0%)! If you want to compare FRAP curves, they all should have been acquired using exactly the same settings, including the amount of photobleaching. Therefore, there is no need at all to do the post-photobleaching normalization, except to mask the differences between different experiments… I know that in many semi-quantitative FRAP articles this is being done, but I would not recommend this since it is physically incorrect. That being said, for small amounts of photobleaching, small differences do not matter so much (see e.g. fig 7 of the Axelrod paper in case of a 2-D spot photobleached by a stationary Gaussian beam).

 

Hope this helps,

 

Best regards,

 

Kevin

 

 

 

Prof. Dr. Kevin Braeckmans

Lab. General Biochemistry and Physical Pharmacy

Ghent University

Harelbekestraat 72

9000 Ghent

Belgium

Tel: [phone]

Fax: [phone]

 

 

 

 

 

 

 

Van: Confocal Microscopy List [[hidden email]] Namens John Runions
Verzonden: dinsdag 18 november 2008 18:37
Aan: [hidden email]
Onderwerp: Laser effects during bleaching

 

Hi Tom,

When doing FRAP, we generally try to keep the bleaching lasers as low as possible so that a loss of fluorescence results but so that we aren't applying enough laser power to damage other proteins in membranes.  If the lasers were so hot as to denature proteins the whole procedure would be invalidated as we'd be killing the cells (I do often imagine the sound of frying bacon during the bleaching phase and hope that it's not really happening!).

At normal laser power levels for confocal, cells seem to function properly over the relatively long term.  The trick during the bleaching phase is to increase the laser power so that a bit of bleaching results.  If the region to be studied is bleached completely black, then I feel there really is the potential for cellular damage.  I've certainly exploded cells with too much laser power so your specimens should be handled gently.  The small amount of bleaching attained using reasonable laser power is analyzed relativistically, i.e. you normalize post-bleach and pre-bleach intensity between 0-100% intensity so that there is no requirement to bleach the fluorescence completely away.  The idea of fluorescence recovery is that any molecule will move to its equilibrium concentration in time (unless constrained).  The species of molecule that we are analyzing the dynamics of in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP can be done, therefore, with a small population of bleached molecules.

In some recent experiments, we have been FRAPing so that only a very small decrease in fluorescence occurs within a region of interest.  Recovery curves fit from these experiments seem equivalent to those fit to data sets in which much more bleaching of the ROI was visible.  We need to ascertain if they are significantly the same, however.  If any physicists of diffusion are listening it would be nice to have your take on this.

Regards, John.

Donnelly, Tom wrote:

Just to add to the confusion.

 

What happens to the non-fluorescent proteins in the cell when you crank up the power to bleach or image in live cells?

 

Tom

-----Original Message-----
From: Confocal Microscopy List [[hidden email]]On Behalf Of John Runions
Sent: Tuesday, November 18, 2008 9:08 AM
To: [hidden email]
Subject: Re: broadband excitation vs. narrow band

Boy, what a stupid question Carl.  I think we should all fail to dignify it with a response!

Actually, that kind of question is fundamental to us in the FRAP world and it sounds like someone needs to do some good empirical measuring.  When bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the argon laser at the same time.  It works better that just using the 488 and I explain this by saying that it is because we are bleaching with a broader spectrum across the excitation range.  I have never been able (or tried) to confirm if this is the case or if the higher power at the specimen plays a role.

Sorry to be not much help.  John.

Carl Boswell wrote:

Hi all,
I was told once that there are no stupid questions, so let's test that assumption.

The question has to do with photobleaching vs. excitation energy.  To get X photons from a fluor, would there be less photobleaching using a single wavelength excitation source at peak excitation wavelength, or a broadband (20-30nm) light source centered on the peak excitation of the molecule, or would there be no difference?  My assumption is that lower "power" (brightness?) would be needed for the broadband source, but would the overall photon flux be greater to get equivalent output?

To take this one step farther, is there less or more bleaching from "inefficient" excitation, i.e. off-peak excitation, to get the same output? If a fluor is less efficiently excited, is it less efficiently bleached, even though more power may be needed to get equivalent output?

Thanks,
Carl

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

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

 

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

 

--

(Sent from my cra%#y non-Blackberry electronic device that still has wires)

 

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions’ lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

[Jonathan M Krupp]

On Tue, 18 Nov 2008 19:46:15 -0700
  John Chandler <[hidden email]> wrote:
> So, Jon,
>
> Which demo samples do you like?  Which ones have good
>effort to results ratio?
>
> Inquiring minds want to know.


I'll get a more comprehensive list together and send it to
you, but for now here's what I have tried.

Simple H&E sections are amazingly useful. I didn't believe
it when someone suggested it, but they really work to demo
fluorescence in a simple way.

I am going to try pollen and some freehand sections of
some local plants asap. Also looks like the prepared test
slides from Invitrogen might be a good bet if you can
afford them.

Jon

[Glen MacDonald-2]

Many aquatic invertebrates have autofluorescence spanning the spectrum  
from UV to red. Also, a DAB immunolabeled tissue slide dipped briefly  
in glutaraldehyde and rinsed before coverslipping will display green-
yellow fluorescence with dark DAB deposits.

Glen
On Nov 19, 2008, at 11:41 AM, Jonathan M Krupp wrote:

> On Tue, 18 Nov 2008 19:46:15 -0700
> John Chandler <[hidden email]> wrote:
>> So, Jon,
>> Which demo samples do you like?  Which ones have good effort to  
>> results ratio?
>> Inquiring minds want to know.
>
>
> I'll get a more comprehensive list together and send it to you, but  
> for now here's what I have tried.
>
> Simple H&E sections are amazingly useful. I didn't believe it when  
> someone suggested it, but they really work to demo fluorescence in a  
> simple way.
>
> I am going to try pollen and some freehand sections of some local  
> plants asap. Also looks like the prepared test slides from  
> Invitrogen might be a good bet if you can afford them.
>
> Jon

[Stephen Cody-2]

G'day List,

I've never done much in the way of deliberate photobleaching
experiments, I'm usually trying to avoid it. But isn't photobleaching
mediated through reactive oxygen species? And if so how can you limit
damage to just the protein you wish to bleach? Or have I missed the
point somewhere?

Cheers
Stephen Cody

2008/11/19 John Runions <[hidden email]>:

> Hi Tom,
>
> When doing FRAP, we generally try to keep the bleaching lasers as low as
> possible so that a loss of fluorescence results but so that we aren't
> applying enough laser power to damage other proteins in membranes.  If the
> lasers were so hot as to denature proteins the whole procedure would be
> invalidated as we'd be killing the cells (I do often imagine the sound of
> frying bacon during the bleaching phase and hope that it's not really
> happening!).
>
> At normal laser power levels for confocal, cells seem to function properly
> over the relatively long term.  The trick during the bleaching phase is to
> increase the laser power so that a bit of bleaching results.  If the region
> to be studied is bleached completely black, then I feel there really is the
> potential for cellular damage.  I've certainly exploded cells with too much
> laser power so your specimens should be handled gently.  The small amount of
> bleaching attained using reasonable laser power is analyzed
> relativistically, i.e. you normalize post-bleach and pre-bleach intensity
> between 0-100% intensity so that there is no requirement to bleach the
> fluorescence completely away.  The idea of fluorescence recovery is that any
> molecule will move to its equilibrium concentration in time (unless
> constrained).  The species of molecule that we are analyzing the dynamics of
> in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP
> can be done, therefore, with a small population of bleached molecules.
>
> In some recent experiments, we have been FRAPing so that only a very small
> decrease in fluorescence occurs within a region of interest.  Recovery
> curves fit from these experiments seem equivalent to those fit to data sets
> in which much more bleaching of the ROI was visible.  We need to ascertain
> if they are significantly the same, however.  If any physicists of diffusion
> are listening it would be nice to have your take on this.
>
> Regards, John.
>
> Donnelly, Tom wrote:
>
> Just to add to the confusion.
>
> What happens to the non-fluorescent proteins in the cell when you crank up
> the power to bleach or image in live cells?
>
> Tom
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]On
> Behalf Of John Runions
> Sent: Tuesday, November 18, 2008 9:08 AM
> To: [hidden email]
> Subject: Re: broadband excitation vs. narrow band
>
>
>
> Boy, what a stupid question Carl.  I think we should all fail to dignify it
> with a response!
>
> Actually, that kind of question is fundamental to us in the FRAP world and
> it sounds like someone needs to do some good empirical measuring.  When
> bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the
> argon laser at the same time.  It works better that just using the 488 and I
> explain this by saying that it is because we are bleaching with a broader
> spectrum across the excitation range.  I have never been able (or tried) to
> confirm if this is the case or if the higher power at the specimen plays a
> role.
>
> Sorry to be not much help.  John.
>
> Carl Boswell wrote:
>
> Hi all,
> I was told once that there are no stupid questions, so let's test that
> assumption.
>
> The question has to do with photobleaching vs. excitation energy.  To get X
> photons from a fluor, would there be less photobleaching using a single
> wavelength excitation source at peak excitation wavelength, or a broadband
> (20-30nm) light source centered on the peak excitation of the molecule, or
> would there be no difference?  My assumption is that lower "power"
> (brightness?) would be needed for the broadband source, but would the
> overall photon flux be greater to get equivalent output?
>
> To take this one step farther, is there less or more bleaching from
> "inefficient" excitation, i.e. off-peak excitation, to get the same output?
> If a fluor is less efficiently excited, is it less efficiently bleached,
> even though more power may be needed to get equivalent output?
>
> Thanks,
> Carl
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology

--
Stephen Cody

[Csúcs Gábor]

Dear Stephen,

I'd argue that photobleaching is a procedure which we barely understand.
Most of the observations/rules are purely empirical and so far I have
seen no comprehensive description of the procedure. Furthermore (as
always) the details may be dependent also on the specific dye/fluorophore.
But back to your question: of course the reactive species created in a
photobleaching experiment will harm also other proteins (or generally
the cell). However, the overall assumption is that your
bleaching/recovery experiment happens in a much faster time scale then
the corresponding cytotoxic effects. Of course there are
processes/methods like CALI (Chromophore Assisted Laser Inactivation)
where people used the "immediate" effect of the putative reactive
species (as far as I know also CALI is a poorly understood procedure).

Greetings    Gabor

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

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

[Craig Brideau]

Here's a related question that's been bugging me:
If photobleaching is due to free radicals, high pulse energy should generate more free radicals and thus more bleaching.
If this is the case, if you need to get more signal from your sample, is it better to increase your pulse energy, or just crank up your average power?

Thoughts?

Craig

On Wed, Nov 19, 2008 at 6:32 PM, Stephen Cody <[hidden email]> wrote:

G'day List,

I've never done much in the way of deliberate photobleaching
experiments, I'm usually trying to avoid it. But isn't photobleaching
mediated through reactive oxygen species? And if so how can you limit
damage to just the protein you wish to bleach? Or have I missed the
point somewhere?

Cheers
Stephen Cody

2008/11/19 John Runions <[hidden email]>:
> Hi Tom,
>
> When doing FRAP, we generally try to keep the bleaching lasers as low as
> possible so that a loss of fluorescence results but so that we aren't
> applying enough laser power to damage other proteins in membranes.  If the
> lasers were so hot as to denature proteins the whole procedure would be
> invalidated as we'd be killing the cells (I do often imagine the sound of
> frying bacon during the bleaching phase and hope that it's not really
> happening!).
>
> At normal laser power levels for confocal, cells seem to function properly
> over the relatively long term.  The trick during the bleaching phase is to
> increase the laser power so that a bit of bleaching results.  If the region
> to be studied is bleached completely black, then I feel there really is the
> potential for cellular damage.  I've certainly exploded cells with too much
> laser power so your specimens should be handled gently.  The small amount of
> bleaching attained using reasonable laser power is analyzed
> relativistically, i.e. you normalize post-bleach and pre-bleach intensity
> between 0-100% intensity so that there is no requirement to bleach the
> fluorescence completely away.  The idea of fluorescence recovery is that any
> molecule will move to its equilibrium concentration in time (unless
> constrained).  The species of molecule that we are analyzing the dynamics of
> in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP
> can be done, therefore, with a small population of bleached molecules.
>
> In some recent experiments, we have been FRAPing so that only a very small
> decrease in fluorescence occurs within a region of interest.  Recovery
> curves fit from these experiments seem equivalent to those fit to data sets
> in which much more bleaching of the ROI was visible.  We need to ascertain
> if they are significantly the same, however.  If any physicists of diffusion
> are listening it would be nice to have your take on this.
>
> Regards, John.
>
> Donnelly, Tom wrote:
>
> Just to add to the confusion.
>
> What happens to the non-fluorescent proteins in the cell when you crank up
> the power to bleach or image in live cells?
>
> Tom
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]On
> Behalf Of John Runions
> Sent: Tuesday, November 18, 2008 9:08 AM
> To: [hidden email]
> Subject: Re: broadband excitation vs. narrow band
>
>
>
> Boy, what a stupid question Carl.  I think we should all fail to dignify it
> with a response!
>
> Actually, that kind of question is fundamental to us in the FRAP world and
> it sounds like someone needs to do some good empirical measuring.  When
> bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the
> argon laser at the same time.  It works better that just using the 488 and I
> explain this by saying that it is because we are bleaching with a broader
> spectrum across the excitation range.  I have never been able (or tried) to
> confirm if this is the case or if the higher power at the specimen plays a
> role.
>
> Sorry to be not much help.  John.
>
> Carl Boswell wrote:
>
> Hi all,
> I was told once that there are no stupid questions, so let's test that
> assumption.
>
> The question has to do with photobleaching vs. excitation energy.  To get X
> photons from a fluor, would there be less photobleaching using a single
> wavelength excitation source at peak excitation wavelength, or a broadband
> (20-30nm) light source centered on the peak excitation of the molecule, or
> would there be no difference?  My assumption is that lower "power"
> (brightness?) would be needed for the broadband source, but would the
> overall photon flux be greater to get equivalent output?
>
> To take this one step farther, is there less or more bleaching from
> "inefficient" excitation, i.e. off-peak excitation, to get the same output?
> If a fluor is less efficiently excited, is it less efficiently bleached,
> even though more power may be needed to get equivalent output?
>
> Thanks,
> Carl
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology



--
Stephen Cody

[David Burk]

GFP Experts:

We have a group transfecting cells with adenovirus.  This virus contains
an EGFP sequence to let you know that, yes, the cell was transfected.
We have noticed in some of the transfected cells that the GFP signal
appears to be strongly associated with lipid droplets.  This is new to
me as I wouldn't expect WT-GFP to strongly associate with any particular
subcellular organelle and is confounding their work since they are
primarily interested in - of course - lipid droplets.

Do any of you know of a reason we would see wild-type GFP with no
targeting sequence or modifications labeling lipid droplets?  We are
beginning some additional control experiments with a large titration
range of virus to determine if the lipid labeling is related to vector
concentration (~GFP expression level).

Thanks for your help!

David H. Burk

[Kevin Braeckmans]

Hi,

 

Not necessarily. Most photobleaching happens from the long-lived triplet state, at least for a fluorescein, a dye that is frequently used for FRAP experiments.

 

First consider the case where a light dose is delivered at a very short time scale. At high laser powers, fluorophores will be quickly pumped to the triplet state, i.e. the triplet state will be saturated.  At that point, photobleaching reactions will compete with relaxation of the triplet state to the ground state (by intersystem crossing). A limited amount of photobleaching results.

 

Now consider the same light dose being delivered by a much reduced light beam over a longer time interval. In this case only a fraction of the fluorophores will in the triplet state at any point in time, but integrated over time, more molecules will have reached the triplet state resulting in more bleaching.

 

We have shown this theoretically as well as experimentally for fluorescein. Hesitating because of the self advertisement, but since the question was raised … details can be found in J Biomed Opt 11(4), 044013 (2006). Specifically you could have a look at Fig 8A.

 

Of course this all depends on the reaction rates of the different photochemical processes taking part in the photobleaching process and might be different for other fluorophores.

 

Hope this helps,

 

Best regards,

 

Kevin

 

Van: Confocal Microscopy List [mailto:[hidden email]] Namens Craig Brideau
Verzonden: donderdag 20 november 2008 17:49
Aan: [hidden email]
Onderwerp: Re: Laser effects during bleaching

 

Here's a related question that's been bugging me:
If photobleaching is due to free radicals, high pulse energy should generate more free radicals and thus more bleaching.
If this is the case, if you need to get more signal from your sample, is it better to increase your pulse energy, or just crank up your average power?

Thoughts?

Craig

On Wed, Nov 19, 2008 at 6:32 PM, Stephen Cody <[hidden email]> wrote:

G'day List,

I've never done much in the way of deliberate photobleaching
experiments, I'm usually trying to avoid it. But isn't photobleaching
mediated through reactive oxygen species? And if so how can you limit
damage to just the protein you wish to bleach? Or have I missed the
point somewhere?

Cheers
Stephen Cody

2008/11/19 John Runions <[hidden email]>:
> Hi Tom,
>
> When doing FRAP, we generally try to keep the bleaching lasers as low as
> possible so that a loss of fluorescence results but so that we aren't
> applying enough laser power to damage other proteins in membranes.  If the
> lasers were so hot as to denature proteins the whole procedure would be
> invalidated as we'd be killing the cells (I do often imagine the sound of
> frying bacon during the bleaching phase and hope that it's not really
> happening!).
>
> At normal laser power levels for confocal, cells seem to function properly
> over the relatively long term.  The trick during the bleaching phase is to
> increase the laser power so that a bit of bleaching results.  If the region
> to be studied is bleached completely black, then I feel there really is the
> potential for cellular damage.  I've certainly exploded cells with too much
> laser power so your specimens should be handled gently.  The small amount of
> bleaching attained using reasonable laser power is analyzed
> relativistically, i.e. you normalize post-bleach and pre-bleach intensity
> between 0-100% intensity so that there is no requirement to bleach the
> fluorescence completely away.  The idea of fluorescence recovery is that any
> molecule will move to its equilibrium concentration in time (unless
> constrained).  The species of molecule that we are analyzing the dynamics of
> in FRAP are the bleached and unbleached versions of the fluorochome.  FRAP
> can be done, therefore, with a small population of bleached molecules.
>
> In some recent experiments, we have been FRAPing so that only a very small
> decrease in fluorescence occurs within a region of interest.  Recovery
> curves fit from these experiments seem equivalent to those fit to data sets
> in which much more bleaching of the ROI was visible.  We need to ascertain
> if they are significantly the same, however.  If any physicists of diffusion
> are listening it would be nice to have your take on this.
>
> Regards, John.
>
> Donnelly, Tom wrote:
>
> Just to add to the confusion.
>
> What happens to the non-fluorescent proteins in the cell when you crank up
> the power to bleach or image in live cells?
>
> Tom
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]On
> Behalf Of John Runions
> Sent: Tuesday, November 18, 2008 9:08 AM
> To: [hidden email]
> Subject: Re: broadband excitation vs. narrow band
>
>
>
> Boy, what a stupid question Carl.  I think we should all fail to dignify it
> with a response!
>
> Actually, that kind of question is fundamental to us in the FRAP world and
> it sounds like someone needs to do some good empirical measuring.  When
> bleaching GFP we will often use all of the 458, 477,488 and 514 lines of the
> argon laser at the same time.  It works better that just using the 488 and I
> explain this by saying that it is because we are bleaching with a broader
> spectrum across the excitation range.  I have never been able (or tried) to
> confirm if this is the case or if the higher power at the specimen plays a
> role.
>
> Sorry to be not much help.  John.
>
> Carl Boswell wrote:
>
> Hi all,
> I was told once that there are no stupid questions, so let's test that
> assumption.
>
> The question has to do with photobleaching vs. excitation energy.  To get X
> photons from a fluor, would there be less photobleaching using a single
> wavelength excitation source at peak excitation wavelength, or a broadband
> (20-30nm) light source centered on the peak excitation of the molecule, or
> would there be no difference?  My assumption is that lower "power"
> (brightness?) would be needed for the broadband source, but would the
> overall photon flux be greater to get equivalent output?
>
> To take this one step farther, is there less or more bleaching from
> "inefficient" excitation, i.e. off-peak excitation, to get the same output?
> If a fluor is less efficiently excited, is it less efficiently bleached,
> even though more power may be needed to get equivalent output?
>
> Thanks,
> Carl
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology
>
> --
>
> (Sent from my cra%#y non-Blackberry electronic device that still has wires)
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology



--
Stephen Cody

 

[mmodel]

Hi David - are you certain those are really lipid droplets and not something like endocytic vesicles?

Mike Model

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Burk
Sent: Thursday, November 20, 2008 2:31 PM
To: [hidden email]
Subject: GFP and lipid localization?

GFP Experts:

We have a group transfecting cells with adenovirus.  This virus contains
an EGFP sequence to let you know that, yes, the cell was transfected.
We have noticed in some of the transfected cells that the GFP signal
appears to be strongly associated with lipid droplets.  This is new to
me as I wouldn't expect WT-GFP to strongly associate with any particular
subcellular organelle and is confounding their work since they are
primarily interested in - of course - lipid droplets.

Do any of you know of a reason we would see wild-type GFP with no
targeting sequence or modifications labeling lipid droplets?  We are
beginning some additional control experiments with a large titration
range of virus to determine if the lipid labeling is related to vector
concentration (~GFP expression level).

Thanks for your help!

David H. Burk

[Mario-2]

I have the same question, and, further, are you sure that the GFP
isn't simply aggregated? If aggregates form they could expose
hydrophobic surfaces that favorably bind to the droplets.

Mario

>Hi David - are you certain those are really lipid droplets and not
>something like endocytic vesicles?
>
>Mike Model
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of David Burk
>Sent: Thursday, November 20, 2008 2:31 PM
>To: [hidden email]
>Subject: GFP and lipid localization?
>
>GFP Experts:
>
>We have a group transfecting cells with adenovirus.  This virus contains
>an EGFP sequence to let you know that, yes, the cell was transfected.
>We have noticed in some of the transfected cells that the GFP signal
>appears to be strongly associated with lipid droplets.  This is new to
>me as I wouldn't expect WT-GFP to strongly associate with any particular
>subcellular organelle and is confounding their work since they are
>primarily interested in - of course - lipid droplets.
>
>Do any of you know of a reason we would see wild-type GFP with no
>targeting sequence or modifications labeling lipid droplets?  We are
>beginning some additional control experiments with a large titration
>range of virus to determine if the lipid labeling is related to vector
>concentration (~GFP expression level).
>
>Thanks for your help!
>
>David H. Burk

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]
[hidden email]

[Christian-103]

Are the "droplets" smooth?  I have seen many different aggregations, some smooth, some look like particles, also what size, roughly are they?

Christian

[David Burk]

1. Q: Hi David - are you certain those are really lipid droplets and not something like endocytic vesicles?

   A: We are pretty convinced they are lipid.  They are counterstained with Bodipy 558/568 and we have localized several "lipid-associated proteins" with them - like TIP47 and ADRP.


2. Q: I have the same question, and, further, are you sure that the GFP isn't simply aggregated? If aggregates form they could expose hydrophobic surfaces that favorably bind to the droplets.

   A = Q:  How would I determine if this were the case?

3. Q:  Are the "droplets" smooth?  I have seen many different aggregations, some smooth, some look like particles, also what size, roughly are they?

   A:  Yep, smooth and round - like nice lipid droplets :)  I don't have the size handy at the moment but can get back to you on that.

Thanks for your input!  I'll be happy to answer more questions if it helps us answer our initial question - quoted below.

David

__________________

GFP Experts:

We have a group transfecting cells with adenovirus.  This virus contains an EGFP sequence to let you know that, yes, the cell was transfected.
We have noticed in some of the transfected cells that the GFP signal appears to be strongly associated with lipid droplets.  This is new to me as I wouldn't expect WT-GFP to strongly associate with any particular subcellular organelle and is confounding their work since they are primarily interested in - of course - lipid droplets.

Do any of you know of a reason we would see wild-type GFP with no targeting sequence or modifications labeling lipid droplets?  We are beginning some additional control experiments with a large titration range of virus to determine if the lipid labeling is related to vector concentration (~GFP expression level).

Thanks for your help!

David H. Burk

[Michael Schell]

The propensity to form smooth, round "aggregates" of GFPs varies greatly among the GFP variants--and it is even more severe with all DsRed derivatives we've used.  In our experience, the "original" EGFP coded by the Clontech vectors is the least prone to forming these structures.  This may be because of a slightly longer N- or C-terminal stretches of amino acids flanking the EGFP sequence, perhaps due to polylinkers in the vector.

We recently compared EGFP to "monster" GFP (also called hmGFP) which is a very bright GFP isolated from sea anemone (expressed in some Promega-derived RNAi vectors, also sold by Superarray).  This GFP variant was far more prone to forming the smooth, round structures that looked like lipid droplets.  These killed our cells if expressed longer than 3 days.  To solve this problem, we had to cut out the hmGFP sequence and replace it with Clontech-derived EGFP.  

Bottom line:  not all EGFPs are the same.  Which one is coded in the adenovirus vector?  Is its sequence truncated relative to the Clontech version of EGFP?  Is is a EGFP from another species besides jellyfish?

Mike

Michael J. Schell, Ph.D., CIV, USUHS
Assist. Professor
Dept. of Pharmacology
Uniformed Services University
4301 Jones Bridge Rd.
Bethesda, MD  20814-3220
tel:  [phone]
[hidden email]
>>> David Burk <[hidden email]> 11/20/08 4:24 PM >>>
1. Q: Hi David - are you certain those are really lipid droplets and not something like endocytic vesicles?

   A: We are pretty convinced they are lipid.  They are counterstained with Bodipy 558/568 and we have localized several "lipid-associated proteins" with them - like TIP47 and ADRP.


2. Q: I have the same question, and, further, are you sure that the GFP isn't simply aggregated? If aggregates form they could expose hydrophobic surfaces that favorably bind to the droplets.

   A = Q:  How would I determine if this were the case?

3. Q:  Are the "droplets" smooth?  I have seen many different aggregations, some smooth, some look like particles, also what size, roughly are they?

   A:  Yep, smooth and round - like nice lipid droplets :)  I don't have the size handy at the moment but can get back to you on that.

Thanks for your input!  I'll be happy to answer more questions if it helps us answer our initial question - quoted below.

David

__________________

GFP Experts:

We have a group transfecting cells with adenovirus.  This virus contains an EGFP sequence to let you know that, yes, the cell was transfected.
We have noticed in some of the transfected cells that the GFP signal appears to be strongly associated with lipid droplets.  This is new to me as I wouldn't expect WT-GFP to strongly associate with any particular subcellular organelle and is confounding their work since they are primarily interested in - of course - lipid droplets.

Do any of you know of a reason we would see wild-type GFP with no targeting sequence or modifications labeling lipid droplets?  We are beginning some additional control experiments with a large titration range of virus to determine if the lipid labeling is related to vector concentration (~GFP expression level).

Thanks for your help!

David H. Burk

[Christian-103]

Michael,

What about the "newer" RFP derived from Entacmaea quadricolor?  Have you seen it aggregate at all?

I guess part of my problem is that I often can not get a hold of which "GFP" I'm being handed, so when there are "punctate spots" or "aggregates" in the cytoplasm, I can only shrug as to what they might be (plant cells by the way).

Christian