Laser effects during bleaching

Posted by John Runions on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Laser-effects-during-bleaching-tp1515193.html

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 [[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]