Re: Photobleaching of GFP in Spinning Disk Confocal

Posted by George Peeters-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wasabi-freeware-tp1574097p1574445.html

Dear Mayandi:

I'm not familiar enough with the "Revolution" to know what 30 means. Most AOTFs can be controlled all the way from essentially off (1/10,000), to some maximum power level. So neutral density filters shouldn't be needed. 240 uW is still quite a bit  of light and I can understand why bleaching may be occurring.  Is "30" as low as you can go?  if you have a CSU-10b it will have an ND built in if you have a CSUX it may or may not have an ND position. What is the other channel (561nm ?).

George

George A. Peeters MD, MS

President,  Solamere Technology Group Inc

1427 Perry Ave

Salt Lake City, UT 84103

www.solameretech.com

801 322-2645 office          801 322-2645 fax

Hi all, One of our client is using a cell line expressing GFP bleaches very fast (Andor Revolution SDC system), despite using low excitation setting at the 488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain. She wants to get two frames per second under two channels she is getting it but the cell bleaches within couple of minutes. Using live cell antioxidants such as Trolox is not an option, I would greatly appreciate any insights in this regard. Will neutral density filters help?

Thanking you in advance
Shiv

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
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