Posted by
leoncio vergara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5038342.html
As it was pointed out in another response, you may need more than 50 iterations to bleach. Also in live cells you may have problems bleaching if the GFP labeled molecule diffuses back into the ROI. You need to bleach faster than the recovery rate (or bleach completely the cell or all comunicating compartments, which may defeat the purpose of a kinetic experiment). This a lot like trying to make a hole in water :)
On a scanning microscope the bleach rate depends not only on the laser power applied but also on the shape and size of the ROI. Since the scan in the x direction is faster compared to the y direction, an ROI oriented with the longer dimension horizontaly will scan faster than another ROI of identical size and shape but oriented with the longest dimension vertically. Remember that when drawing the ROIs to minimize the bleaching time.
Also rmemeber that the scan region is always rectangular no matter what the shape of the ROI. If you draw an elliptical or free shape ROI, the scanned area will always be the circumscribed rectangle, for pixels outside the ROI but inside that rectangle, the AOTF will will simply block the light but the pixel will still be scanned.
Once you setup the parameters and the ROI, use the "test bleach" button in the Bleach settings window to test if the bleach works before running your time lapse experiment (in another cell of course).
Hope this helps
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of G.Liu
Sent: Tuesday, May 11, 2010 4:04 PM
To:
[hidden email]
Subject: Re: Photobleach (FRAP) problem in Zeiss 510Meta
The sample is tobacco leaf (live).
And I have contacted Zeiss specialist. Due to no internet hookup for the confocal computer, I did not expect a remote LIVE online trouble shooting.
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