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
	
	

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	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
	
	
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	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
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	>Berkeley, CA
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