Re: Laser effects during bleaching

Posted by John Runions on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Laser-effects-during-bleaching-tp1515193p1515280.html

Hi again Tom, I also meant to say in the last email that I sent you that bleaching of a fluorescent molecule should result because it loses electrons irreversibly during repeated rounds of excitation and emission - not because it is damaged or it's structure is altered in any way by the laser power used.  If that is the case, then there shouldn't be any damage to any other proteins either.  This is a difficult proposition to prove so we usually operate on the assumption that if the membrane or organelle being bleached recovers and the cell continues top live happily then the laser power was below damaging levels.  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 [[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 [phone]