Hi,
In fact, generally speaking, this is not the case. For example,
the recovery half-time generally depends on the amount of photobleaching. There
is one exception: the photobleaching of a 2-D uniform disk. This is clearly
explained in the ‘father of all FRAP articles’: Axelrod et al 1976,
Biophys J 16.
For the same reason I strongly discourage the normalization to
the post-photobleaching level (i.e. putting the post-photobleaching level to
0%)! If you want to compare FRAP curves, they all should have been acquired
using exactly the same settings, including the amount of photobleaching. Therefore,
there is no need at all to do the post-photobleaching normalization, except to
mask the differences between different experiments… I know that in many semi-quantitative
FRAP articles this is being done, but I would not recommend this since it is
physically incorrect. That being said, for small amounts of photobleaching,
small differences do not matter so much (see e.g. fig 7 of the Axelrod paper in
case of a 2-D spot photobleached by a stationary Gaussian beam).
Hope this helps,
Best regards,
Kevin
Prof. Dr. Kevin Braeckmans
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89
Van: Confocal Microscopy List
[mailto:[hidden email]] Namens John Runions
Verzonden: dinsdag 18 november 2008 18:37
Aan: [hidden email]
Onderwerp: Laser effects during bleaching
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