http://confocal-microscopy-list.275.s1.nabble.com/Laser-effects-during-bleaching-tp1515193p1555571.html
experiments, I'm usually trying to avoid it. But isn't photobleaching
> 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 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:
[hidden email]
> 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:
[hidden email]
> 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