Re: Photobleach (FRAP) problem in Zeiss 510Meta

Posted by John Runions on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5040133.html

Hi Guosheng,

We have been doing quite a lot of FRAP and photoactivation in tobacco leaves and I just want to add a couple of things to what the others have said.  If you are using the older software (pre-ZEN), one small thing that seems to cause the 510 Meta software to get a bug in its brain is the difference between the ROI controller and the Bleach Region controller windows.  If you set the ROI for bleaching using the Edit ROI window (accessed from the main LSM window) rather than the Bleach Region window, the bleaching laser won't fire.  This is a rather commonplace confusion as the two windows look virtually identical.

Secondly, what tissue / organelle are you trying to bleach?  GFP in the plasma membrane usually bleaches very well indeed leaving a nicely defined image of the ROI but GFP in the ER or cytoplasm FLIPs (Fluorescence Loss in Photobleaching).  The cytoplasm and ER are such dynamic systems that during your 50-100 bleaching pulses all of the GFP in the region around the ROI moves through the ROI with the effect of reducing overall GFP intensity in the cell rather than producing a nicely bleached ROI shape.

My experience with this system is that you won't need anything like 50-100 bleaching scans.  Especially since you have indicated that you have a 405 laser (which is wicked).  Once you have the system set up properly, you should be able to bleach GFP in tobacco cells with very few pulses indeed.  With a 63x objective, we do it with 2-10 iterations of 405 laser set at 25-50% transmission.

Your results may vary!

Please write to me if you have any more questions about this.

All the best, John.

G.Liu wrote:

Dear all,

We have encountered a problem in our 510Meta for photobleaching. We simply
could not get the GFP ROI bleached. What we did is as following, anything
wrong or inappropriate?


1. Collect the Single unsaturated image as usual (normal settings).

2. Go to Bleach control for bleach settings. Select xx scans before the
bleach. For GFP, tried 1-50 iteration, defined the Bleach region. All the
argon laser lines (458, 488, 514) or only 488 to 100% . 

3. Go to Time Lapse--using Manual start, manual stop (input the number of
scan). Time interval 0 or 1 sec.

4. Hit StartB. 

I can get a series of images but no bleaching was found. The other odd thing
is that "Bleach" button seems not not working--if hit, it only lasts a few
seconds then stop. I've tried using diode405 line, it worked once, but I
can't repeat it anymore.

Any suggestions or advice are greatly appreciated!!

Guosheng