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