A recent paper by Hell et al argues that bleaching is increased by a second photon hitting a molecule that is in the triplet state.
They demonstrate that pulsed exictation, when the interval between pulses is sufficient for molecules to escape from the triplet state, produces greater fluorescence for a given amount of bleaching.
 
 
 
 
 
Major signal increase in fluorescence microscopy through dark-state relaxation 

Gerald Donnert, Christian Eggeling, Stefan W Hell

SUMMARY: We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 ?s, such as the triplet 

Nature Methods 4, 81 - 86 (01 Jan 2007), 

 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories F5
Stockholm University
Stockholm 106 91
Sweden

________________________________

From: Confocal Microscopy List on behalf of Kevin Braeckmans
Sent: Thu 20-Nov-08 20:37
To: [log in to unmask]
Subject: Re: Laser effects during bleaching



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:[log in to unmask]] Namens Craig Brideau
Verzonden: donderdag 20 november 2008 17:49
Aan: [log in to unmask]
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 <[log in to unmask]> 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 <[log in to unmask]>:
> 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:[log in to unmask]]On
> Behalf Of John Runions
> Sent: Tuesday, November 18, 2008 9:08 AM
> To: [log in to unmask]
> 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 520-621-3709
>
> --
>
> (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:  [log in to unmask]
> phone: +44 (0) 1865 483 964
>
> 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:  [log in to unmask]
> phone: +44 (0) 1865 483 964
>
> Runions' lab web site
>
>
>
> Visit The Illuminated Plant Cell dot com
> Oxford Brookes Master's in Bioimaging with Molecular Technology



--
Stephen Cody