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Joost,
If you are using only 4% - 8% laser power for bleaching, how long is your bleaching time? Just beware that the bleaching time should not exceed 10% (maximum!) of the characteristic diffusion time, which is calculated from
R^2/(4*D)
where R is the radius of the bleached spot, and D the diffusion coefficient.
Good luck with your experiments,
Kevin
> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List
> [mailto:[log in to unmask]]Namens Willemse, Joost
> Verzonden: zondag 12 oktober 2003 15:05
> Aan: [log in to unmask]
> Onderwerp: Re: FRAP
>
>
> Generally I think FRAP studies tend to use a bit too high laser
> power. I try to bleach at least 25 % of my fluorophore in my ROI
> and I manage to do that with about 4% -8% laser power whereas for
> scanning i use only 1 percent.
>
> This seems to me that this could hardly influence the state of
> the cell in any way because half of the people use even higher
> laser powers just to make images and all these images would then
> not be realiable either
>
>
>
> -----Oorspronkelijk bericht-----
> Van: Scott Snyder [mailto:[log in to unmask]]
> Verzonden: vr 10/10/2003 3:31
> Aan: [log in to unmask]
> CC:
> Onderwerp: Re: FRAP
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Here is a question I have been wanting to bring up. Is a
> reaction with
> triplet oxygen the only way to quench an XFP? I know that
> for the excited
> state to cross over to a state where it can fluoresce, it
> must release
> energy as heat. Can it release enough heat to denature
> itself and possibly
> proteins around it? I know with FRAP many of the
> photobleachings tend to
> use a ton of laser power to ensure complete bleaching.
> This seems like it
> could lead to problems with thermal denaturation. It could
> also lead to a
> discrepancy in results if one person pours a lot more laser
> power into their
> bleach area than another person does.
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]]On
> Behalf Of Guy Cox
> Sent: Friday, October 10, 2003 1:07 AM
> To: [log in to unmask]
> Subject: Re: FRAP
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I'm not suggesting that depleting oxygen will be sufficient to
> prevent reactive oxygen species forming IF something is forming
> them. What I'm questioning is whether a FRAP bleach of (eg)
> FITC will create any reactive oxygen species. I can't see why
> it would. Bleaching is probably just the reaction of fluorescein
> with normal molecular oxygen, via the mechanism I gave.
>
> Guy
>
>
>
> >>>While I agree with Jim in principle on this one, I would make a
> >>>coupl of comments.
> >>>
> >>>1. Bleaching is most often a result of electron being
> excited into
> >>>a triplet state which will then react very easily with
> oxygen (which is
> >>>naturally in a triplet state) since triplet-triplet reactions are
> >>>favoured. Hence bleaching will deplete oxygen not the opposite.
> >>>Antifades (= photographic developers) are mild reducing
> agents which
> >>>scavenge oxygen to prevent bleaching.
> >
> >I would qualify the above statement. Depleting oxygen in the manner
> >described is insufficient to prevent radical cascade as
> alluded to in
> >my previous posting. As a rule, the only way to kill a radical is
> >with another radical. Some antifades work by either
> forming a poorly
> >reactive radical species, or as in the case of cysteine, self
> >terminates by combining with itself to form cystine. In a membrane,
> >once you've got a carbon centered radical, hope for the nearest
> >Vitamin E.
> >--
> >_________________________________________________________________
> >Mario M. Moronne, Ph.D.
> >NanoMed Technologies LLC
> >President and CTO
> >ph (510) 528-2400
> >FAX (510) 528-8076
> >1561 Posen Ave
> >Berkeley, CA
> >94706
> >
> >[log in to unmask]
> >[log in to unmask]
>
>
> Assoc. Prof. Guy Cox, ooOOOOOOoo
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